Gyrb and Rpob Genes

DIVERSITY AMONG BACTERIAL SYMBIONTS OF ENTOMOPATHOGENIC NEMATODES

HEATHER SMITH KOPPENHÖFER

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2017

In memory of my mother, Linda Huffman, who always believed in me, and to my husband, Albrecht, and our daughter, Katharina. Your encouragement and faith in me has helped make this possible.

ACKNOWLEDGMENTS

I am deeply grateful for the generosity shown to me by many people. I express my sincere gratitude and appreciation to Dr. Frank Louws (North Carolina State University) who shared his expertise in bacterial diversity and allowed me to conduct all of the rep-PCR portion of my dissertation in his laboratory; to Dr. Susan Webb, who allowed me to use equipment in her laboratory; to Dr. Oscar Liburd, who helped provide reagents; to Drs. John Capinera and

William B. Crow, who provided a source of funding for sequencing reactions; and to Dr. Pauline

Lawrence, who provided reagents when I had none and took the time to be a mentor to me even though I was not her student. I thank Dr. Randy Gaugler (Rutgers University) for his advice, for providing funding for meetings and for providing me with a quiet office where I could write. I also thank Dr. Michael Klein (The Ohio State University) who provided funding to attend an important meeting in my field and encouragement to not give up, and Dr. Jessica Ware (Rutgers

University), who always had an answer to my phylogenetic questions. I would like to thank Dr.

Erica Goss for her help with bacterial recombination, and also I would to thank Dr. Matt

Gizendanner and Dr. Mark Miller (San Diego Supercomputer Center) for their assistance with

HiPerGator and Cipres, respectively. I am thankful for technical assistance from Dr. Khuong

Nguyen, Ms. Ellen Dickstein and Mr. Jerry Minsavage. I am grateful to Drs. James and

Alejandra Maruniak who opened their lab to me and helped me with various molecular techniques. I am also grateful to Dr. Jim Maruniak for never giving up on me. It is because of his encouragement that I continued this work. I am grateful to Dr. Jeff Jones who took me on as his student though I was from another department, provided laboratory space, directed me to people with different areas of expertise who could help with this project and guided my work.

I would like express my deep appreciation for Mrs. Debbie Hall, who not only stayed on top of my program more than I but became a great friend in the process. I am especially grateful

for Dr. Heather McAuslane and Ms. Ruth Brumbaugh and all the work they have done so that I could be readmitted into the program. I would like to thank my committee members Dr. James

Preston and Dr. David Reed and past committee members Dr. Steve Forst and Dr. Byron Adams for their time, support and encouragement. The research presented her was started under the direction of Dr. Adams. I am most thankful to my chair and co-chair Dr. Jeffrey B. Jones and

Dr. James E. Maruniak. Their guidance has been invaluable.

I am very grateful for all my wonderful friends and family. I could not have done this without them. Finally, I am especially thankful for Dr. Albrecht Koppenhöfer (Rutgers

University), who provided financial support for much of this research, shared his expertise in entomopathogenic nematodes, discussed ideas with me and helped edit my writing. My husband’s strength, generosity and humility continue to amaze me. This dissertation is an acknowledgement of God’s wondrous creation and the privilege of studying it.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF FIGURES ...... 10

ABSTRACT ...... 12

CHAPTER

1 GENERAL INTRODUCTION ...... 14

Xenorhabdus and Photorhabdus Life Cycle...... 15 Phenotypic Variation ...... 17 Taxonomy and Systematics of Xenorhabdus and Photorhabdus ...... 19 Xenorhabdus and Photorhabdus Taxonomy ...... 22 The Genus Xenorhabdus Thomas & Poinar, 1979 ...... 24 Characters of Xenorhabdus ...... 25 The Genus Photorhabdus Boemare, Akhurst & Mourant, 1993 ...... 26 Characters of Photorhabdus ...... 27 Nematode/Bacteria Associations ...... 29 Phylogenetic Systematics ...... 35 Coevolution ...... 37 Aims and Objectives ...... 38

2 DIVERSITY OF Photorhabdus SPP. ISOLATED IN FLORIDA ...... 46

Introduction ...... 46 Materials and Methods ...... 50 Collection of Nematode Specimens and Isolation of Bacterial Strains ...... 50 Preparation of Genomic DNA ...... 52 Repetitive Sequence-based Genotyping ...... 52 Amplification, cloning and sequencing of partial gyrB and rpoB genes ...... 53 Phylogenetic Analyses ...... 56 Recombination Analyses ...... 59 Results...... 60 Isolation of nematodes ...... 60 Diversity as Assessed by rep-PCR ...... 60 gyrB and rpoB based Phylogenetic Analyses ...... 62 Recombination ...... 64 Discussion ...... 68

3 GENETIC DIVERSITY AMONG Xenorhabdus SPP. AND THEIR PHYLOGENETIC INFERENCE ...... 101

Introduction ...... 101

Materials and Methods ...... 103 Collection of Nematode Specimens, Isolation of Bacterial Strains and Growth Conditions ...... 103 Preparation of Genomic DNA ...... 104 Repetitive Sequence-based Genotyping ...... 105 Gene Selection and Amplification ...... 106 The gyrB Sequence ...... 107 The recA Sequence ...... 107 The groEL Sequence ...... 107 The rpoB Sequence ...... 108 The 16S rDNA Sequence ...... 108 Cloning and Sequencing ...... 109 Alignments ...... 111 Phylogenetic Analyses ...... 111 Recombination Analyses ...... 113 Results...... 113 Diversity as Assessed by rep-PCR ...... 113 Phylogenetic Analyses ...... 115 Recombination ...... 119 Discussion ...... 120

4 A STUDY OF THE 16 S rRNA IN Xenorhabdus spp...... 145

Introduction ...... 145 Materials and Methods ...... 146 Isolation of Bacterial Strains ...... 146 Preparation of Genomic DNA ...... 146 The 16S rDNA Sequence ...... 147 Alignments ...... 148 Results...... 150 Discussion ...... 152

5 CLOSING REMARKS ...... 165

APPENDIX

A rRNA SEQUENCES ...... 169

B FATTY ACID METHYL ESTER COMPOSITION ...... 171

LIST OF REFERENCES ...... 177

BIOGRAPHICAL SKETCH ...... 202

LIST OF TABLES Table page

1-1 Symbiotic Associations ...... 40

2-1 Photorhabdus strains and form variants used for this study ...... 77

2-2 DNA primers used for rep-PCR ...... 79

2-3 Strains and GenBank accession numbers used for phylogenetic analyses ...... 80

2-4 Models of nucleotide substitutions selected for gyrB and rpoB datasets...... 82

2-5 Pairwise homoplasy index test for recombination ...... 83

2-6 Intragenic recombination events as detected using GARD ...... 83

2-7 Putative recombination events in gyrB, rpoB and gyrB-rpoB concatenated sequences detected by RDP4 ...... 84

2-8 Pairwise homoplasy index test for recombination of portions of gene sequences suspected of recombination events ...... 85

3-1 Xenorhabdus strains used for this study ...... 128

3-2 Sequences of primers used to amplify and sequence gene regions ...... 130

3-3 GenBank accession numbers for additional gene sequences used in the present study ..131

3-4 Pairwise homoplasy index test (PHI) and Genetic algorithm recombination detection (GARD) to screen for recombination ...... 133

4-1 Bacterial isolates used for this study and number of 16S rRNA copies ...... 157

4-2 Sequences of primers used to amplify and sequence gene regions ...... 158

4-3 GenBank accession numbers for additional 16S gene sequences used in this study ...... 159

4-4 Similarity values for the 16S rRNA sequence for the two undescribed Xenorhabdus strains to known species and to each other ...... 160

B-1 X. nematophila, ATCC 39497 ...... 171

B-2 X. szentirmaii, 17C ...... 172

B-3 X. koppenhoeferi, AMK001 ...... 173

B-4 X. koppenhoeferi, AMK002 ...... 173

B-5 X. bovienii, XbINT (S. intermedium) ...... 174

B-6 P. temperata (H. megidis) ...... 175

B-7 P. heterorhabditis, ENY25 ...... 175

B-8 P. luminescens subsp. laumondii, (H. bacteriophora, HP88) ...... 176

LIST OF FIGURES

Figure page

1-1 16S rRNA gene-based phylogeny for relationships of genera within the family Enterobacteriaceae ...... 42

1-2 Phylogenetic relationships of Xenorhabdus based on maximum likelihood analysis of partial 16S rDNA sequences ...... 43

1-3 Phylogenetic relationships of Photorhabdus based on maximum likelihood analysis of partial 16S rDNA sequences...... 44

1-4 Phylogenetic relationships between species/strains of Photorhabdus based on maximum likelihood analysis of partial gyrB gene sequences ...... 45

2-1 Sampling sites containing entomopathogenic nematodes from which bacteria were isolated for this study ...... 86

2-2 Cluster analysis of banding patterns of Photorhabdus strains ...... 87

2-3 Nucleotide variability of gyrB and rpoB gene sequences of Photorhabdus strains ...... 88

2-4 Phylogenetic tree from ML analysis of gyrB partial gene sequences for 48 Photorhabdus strains ...... 89

2-5 Phylogenetic relationships between species/strains of Photorhabdus based on maximum likelihood analysis of partial rpoB gene sequences ...... 90

2-6 Comparsion between the gyrB gene rpoB gene Bayesian trees ...... 91

2-7 Phylogenetic relationships between species/strains of Photorhabdus based on maximum likelihood analysis of concatenated gyrB-rpoB partial gene sequences ...... 92

2-8 Split decomposition graphs for P. luminescens strains and P. asymbiotica, P. heterorhabditis and P. temperata strains ...... 93

2-9 Split decomposition graph for Photorhabdus strain using rpoB partial DNA sequences...... 94

2-10 Split decomposition graph for Photorhabdus strains using concatenated gyrB-rpoB partial DNA sequences ...... 95

2-11 Alternative tree topologies given by using different regions the gyrB partial gene sequence ...... 96

2-12 Position of P. heterorhabditis Q614, P. temperata subsp. cinerea and P. luminescens subsp. kleinii as determined by region of gyrB gene used for phylogenetic reconstruction...... 97

2-13 Alternative tree topologies for Photorhabdus based upon different regions of the rpoB gene...... 98

2-14 Alternative tree topologies for Photorhabdus based upon different regions of the rpoB gene ...... 99

2-15 Photorhabdus maximum likelihood tree of concatenated gyrB-rpoB sequences with potential regions of recombination removed ...... 100

3-1 Circular genomic map of X. nematophila with positions of genes used in this study. ....134

3-2 Regions of genes selected for this study with locations of PCR and sequencing primers...... 135

3-3 Cluster analysis of banding patterns of Xenorhabdus strains ...... 136

3-4 Phylogenetic relationships between species/strains of Xenorhabdus based on Bayesian analysis of partial gyrB and recA sequences...... 137

3-5 Xenorhabdus maximum likelihood tree and Bayesian majority rule consensus tree of the combined gyrB and recA sequences ...... 138

3-6 Bayesian analysis of the groEL and rpoB partial sequences for Xenorhabdus, majority rule consensus trees ...... 139

3-7 Phylogenetic analyses of the combined gyrB, recA, groEL and rpoB sequences for Xenorhabdus ...... 140

3-8 Maximum parsimony analysis of Xenorhabdus 16S rDNA partial sequences...... 141

3-9 Phylogenetic relationships between species/strains of Xenorhabdus based on maximum likelihood analysis of partial 16S rRNA ...... 142

3-10 Split decomposition graph of concatenated housekeeping genes for Xenorhabdus strains ...... 143

3-11 Maximum likelihood tee of Xenorhabdus concatenated gyrB-recA-rpoB-groL gene sequences with putative recombination sites removed ...... 144

4-1 Regions where Xenorhabdus 16S rRNA gene sequences were ambiguously aligned ..161

4-2 Maximum Parsimony tree based on Xenorhabdus 16S rRNA sequences ...... 162

4-3 Bayesian analysis of Xenorhabdus using 16S rRNA sequences ...... 163

4-4 Split decomposition graph of Xenorhabdus based on 16S rRNA...... 164

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

DIVERSITY AMONG BACTERIAL SYMBIONTS OF ENTOMOPATHOGENIC NEMATODES

Heather Smith Koppenhöfer

May 2017

Chair: Jeffrey B. Jones Cochair: James E. Maruniak Major: Entomology and Nematology

Nematodes of the family Steinernematidae and Heterorhabditidae are obligate insect pathogens that share a mutualistic association with bacteria of the genera Xenorhabdus and

Photorhabdus, respectively. The infective juvenile stage of the nematode harbors cells of the bacterial symbiont within its intestinal tract. Following entry of the nematode into the hemocoel of an insect host, the bacteria are released and proliferate, killing the insect and providing nutrients that optimize nematode growth and reproduction. Because the nematode-bacterium complex is used extensively for the biological control of insect pests, many different aspects beyond their application as biocontrol agents are being studied including their ecology, biology, coadaptation and coevolution. As the number of known nematode species has been increasing steadily, there is a growing interest in the diversity of their bacterial symbionts. In this study,

Heterorhabditis nematodes were isolated throughout central Florida in areas adjacent to productive citrus groves, and intrageneric relationships within Photorhabdus spp. isolated from these nematodes were investigated. Genomic fingerprints were generated based on the amplification of repetitive DNA sequences distributed throughout the bacterial chromosome

(rep-PCR) (BOX element, repetitive palindromic [REP], and enterobacterial repetitive intergenic

consensus [ERIC]). Unique fingerprints were produced for each bacterial strain separating the strains into species and subspecies thereof. Representative strains were selected for phylogenetic analyses using partial nucleotide sequences for gyrB and rpoB, confirming the rep-PCR results.

Two species of bacteria were identified in Florida, including P. luminescens (two subspecies) and the first report of P. heterorhabditis. To determine the genetic diversity of Xenorhabdus isolated from nematodes throughout the world, rep-PCR was conducted generating genomic fingerprints that formed consistent groups corresponding to bacterial species. From these groups, representative strains were selected for phylogenetic analyses using four housekeeping genes (gyrB, groEL, recA and rpoB) and the 16S rRNA gene. The gene sequences were used individually revealing topological differences among the phylogenetic trees. The protein-coding genes were concatenated and provided the most robust phylogeny. The data obtained for

Photorhabdus and Xenorhabdus suggested that genes for both have undergone lateral gene transfer, which points to the importance of using multiple genes when investigating their evolutionary histories.

CHAPTER 1 GENERAL INTRODUCTION

Bacteria of the genera Xenorhabdus Thomas & Poinar, 1979 and Photorhabdus Boemare,

Akhurst & Mourant, 1993 share a mutualistic relationship with nematodes of the families

Heterorhabditidae and Steinernematidae, respectively. The bacteria are Gram-negative, nonfermentative rods, belonging to the family Enterobacteriaceae. Photorhabdus and

Xenorhabdus form a unique group being phenotypically (Holt et al., 1994) and genotypically

(Brenner & Farmer, 2005) similar to no other genera within the family. However, they both produce the enterobacterial common antigen, which is universally present among the species of

Enterobacteriaceae (Ramia et al., 1982). Generally, the bacterial symbionts are pathogenic to insects, and one species, Photorhabdus asymbiotica Fischer-Le Saux, Viallard, Brunel, Normund

& Boemare, 1999, has been identified as an opportunistic pathogen to humans (Farmer et al.,

1989; Peel et al., 1999). The bacteria are transported by their associated nematodes into the hemocoel of a host insect and, once inside, the nematodes use their symbiotic bacteria to overcome the immune system of the host. The bacteria proliferate, release endo- and exotoxins, septicemia ensues, and insect death generally occurs within 1-3 days (Poinar, 1990; Forst &

Clarke, 2002). The bacteria support nematode growth and reproduction by providing nutrients, antimicrobial agents that inhibit the growth of other organisms, including bacteria, yeast and fungi (Poinar & Thomas, 1966; Akhurst & Boemare, 1988), and insect deterrent factors that protect the nematodes from scavenging insects (Zhou et al., 2002). The nematodes produce up to three generations within the host and, once the host cadaver is depleted, a new cohort of infective juveniles (IJ) exits into the environment taking their respective symbionts with them.

Several of the symbiotic bacteria have been screened and assayed for their insecticidal properties, and their virulence factors have been well studied. Less research has been directed

toward elucidating the taxonomy, systematics and phylogeny of these bacteria. Inter- and intrageneric relationships within Photorhabdus and Xenorhabdus have been analyzed revealing considerable diversity among the symbionts. Some 14 new species of Xenorhabdus (Lengyel et al., 2005; Tailliez et al., 2006) and three new subspecies of Photorhabdus (Akhurst et al., 2004;

Hazir et al., 2004) have been described since 2004, but many bacterial isolates from described nematode species have yet to be examined.

Xenorhabdus and Photorhabdus Life Cycle

Nematodes of the families Heterorhabditidae and Steinernematidae are obligate insect pathogens in nature and are only able to persist in the environment outside a host as the specialized third-stage infective juvenile (IJ). Their bacterial symbionts are located within the intestinal tract of the IJ. In Heterorhabditis, Photorhabdus primarily colonizes the anterior region of the intestine just posterior to the basal bulb and, to varying degrees, is also located throughout the remainder of the intestine (Endo & Nickle, 1991; Ciche & Ensign, 2003).

Steinernema species have a specialized bilobed intestinal vesicle that is colonized by

Xenorhabdus (Bird & Akhurst, 1983; Martens et al., 2003). In Steinernema carpocapsae, the intestinal vesicle is formed by two perivesicular cells, and a subcellular structure, the IVS

(intravesicular structure), is contained within the lumen unattached to the vesicle (Martens &

Goodrich-Blair, 2005). The IVS and the colonizing Xenorhabdus nematophila (Poinar &

Thomas, 1965) Thomas & Poinar, 1979 co-localize and this IVS-bacterial interaction may be responsible for specific and efficient colonization of the IJ (Martens & Goodrich-Blair, 2005). In both nematode-symbiont systems, the bacteria persist in a quiescent state while within the IJ.

However, limited bacterial growth occurs in S. carpocapsae until the intestinal vesicle is colonized by X. nematophila (Martens et al., 2003).

Once the IJ gains entry into the hemocoel of a suitable insect host, the nematode recovers from developmental arrest and liberates its bacterial symbiont. However, S. carpocapsae was found to recover when in contact with insect gut content prior to moving into the hemocoel

(Sicard et al., 2004a). While inside the insect gut, X. nematophila cells are released from the vesicle into the nematode intestine (Sicard et al., 2004a) and, as the nematode enters the hemocoel, the bacteria are released by defecation (Poinar & Thomas, 1966; Wouts, 1984;

Martens et al., 2004; Sicard et al., 2004a). Photorhabdus exits through the mouth of the nematode in a manner similar to regurgitation (Ciche & Ensign, 2003).

The bacteria and nematodes cooperate to overcome the host’s immune response, allowing the bacteria to proliferate vegetatively (see Dowds & Peters, 2002). Steinernema carpocapsae is able to suppress the immune response by secreting proteins, which may facilitate the release of their symbionts (Götz et al., 1981; Simões, 1998). It is unknown if similar proteins are produced by Heterorhabditis (Forst & Clarke, 2002). In Manduca sexta, Photorhabdus luminescens

(Thomas & Poinar, 1979) Boemare, Akhurst & Mourant, 1993 cells secrete an antiphagocytic factor that allows the bacterial cells to impede their own phagocytosis (Silva et al., 2002). In

Spodoptera exigua, X. nematophila cells are able to hamper nodule formation by inhibiting the eicosanoid biosynthetic pathway (Park & Kim, 2000; Park et al., 2003). Furthermore, X. nematophila inhibits transcription of insect genes encoding antimicrobial peptides in S. exigua and M. sexta (Ji & Kim, 2004; Park et al., 2007). Photorhabdus luminescens and X. nematophila release lipopolysaccharide (LPS) or endotoxin, which is a component of the cell outer membrane

(Dunphy & Thurston, 1990). X. nematophila LPS inhibits prophenoloxidase activity (Dunphy &

Webster, 1991) and in both systems the lipid A moiety is thought to be cytotoxic to hemocytes

(Dunphy & Webster, 1988, 1991). Both genera produce hemolysin activity (Brillard et al., 2001,

2002). The bacteria proliferate within the insect host, while producing toxins and exoenzymes that result in septicemia and bioconversion of the cadaver and provide nutrition for the developing nematodes (see Forst & Clarke, 2002). Early in infection, and prior to insect death,

X. nematophila cells specifically proliferate extracellularly in the hemolymph and connective tissues surrounding the midgut in Spodoptera littoralis (Sicard et al., 2004a). Similarly, in M. sexta, Photorhabdus proliferates in the hemocoel destroying the immune system, and in the midgut where the bacteria release toxins and, late in infection, a metalloprotease that destroys the midgut epithelium and may facilitate bioconversion of the tissue (Bowen et al., 1998; Silva et al., 2002). Additionally, the Photorhabdus mcf (makes caterpillars floppy) gene encodes a toxin that also destroys hemocytes and the insect midgut (Daborn et al., 2002).

Near the end of bacterial growth, the symbionts produce a variety of antimicrobial compounds that protect the cadaver from colonization by other organisms. Such compounds include bacteriocins that are active against closely related bacteria (Thaler et al., 1995; Sharma et al., 2002). Other compounds produced by the bacteria are antibiotics that are active against other bacteria, fungi and yeasts (Akhurst, 1982; Boemare et al., 1997a; Webster et al., 2002).

The developing nematodes feed on the bacteria and bioconverted host tissue and reproduce in the cadaver for one to three generations until the food resources in the cadaver are exhausted. The nematodes develop a new generation of IJ that acquire symbiotic bacterial cells and emerge from the host cadaver in search of a new host.

Phenotypic Variation

Both Xenorhabdus and Photorhabdus produce phenotypic variant forms a feature that has been referred to as phase variation (Boemare & Akhurst, 1988; Forst & Clarke, 2002). The primary form (form I, phase I) is found naturally associated with the nematode, while the variant or secondary form (form II, phase II) spontaneously arises when the bacteria are in culture or

occasionally in the cadaver of the insect during late stages of nematode reproduction (Akhurst,

1980). The two forms differ morphologically and physiologically.

Form I cells are motile by peritrichous flagella (usually absent in form II) and are larger than form II cells (Forst et al., 1997). Form I cells are generally able to adsorb certain dyes, form crystalline inclusion bodies, produce antibiotics, lipase, protease, and in Photorhabdus, some strains bioluminesce (Akhurst, 1980, 1982; Couche et al., 1987; Boemare & Akhurst,

1988; Forst et al., 1997). These characteristics are greatly reduced or lost in the variant cells, and in X. nematophila the secondary form does not produce OpnB, a stationary phase outer membrane protein (Volgyi et al., 2000). Other traits, which tend to be primary form-specific in many but not all strains, include the cell’s ability to lyse red blood cells, to be motile and to produce fimbriae and pigment (Akhurst, 1980; Givaudan et al., 1995; Forst et al., 1997; Volgyi et al., 1998). On nutrient agar Xenorhabdus form II colonies have little or no pigment and

Photorhabdus form II have differential pigmentation that is strain/species-dependent (Akhurst,

1983a; Boemare & Akhurst, 1988; Boemare et al., 1997b).

The secondary variants of both X. nematophila and P. luminescens have higher levels of respiratory enzyme activity, increased membrane potential and, after a period of starvation, are able to commence growth more quickly than their primary form counterparts, indicating that form II cells are able to take in nutrients more efficiently (Smigielski et al., 1994). Pathogenicity is maintained in form II but differences in pathogenicity between phenotypic variants of X. nematophila have been reported in lepidopteran hosts. Form II variants differ in pathogenicity on M. sexta, with being avirulent (Volgyi et al., 1998, 2000), whereas form I and form II are equally pathogenic against G. mellonella (Akhurst, 1980). Form II of Photorhabdus does not support growth and reproduction of Heterorhabditis spp. (Gerritsen & Smits, 1993, 1997).

Conversely, X. nematophila form II cells have the ability to support S. carpocapsae reproduction in vitro (Ehlers et al., 1990; Volgyi et al., 2000) and in vivo (Sicard et al., 2005).

When S. carpocapsae was provided with an equal quantity of both cell variants, form II was almost always retained by the nematode (Sicard et al., 2005). Form II cells allowed for equal rates of reproduction of the nematode and provided protection for the nematode, specifically against antagonistic bacteria sensitive to xenorhabdicin (Sicard et al., 2005). The production of xenorhabdicin is maintained in the secondary variant. However; when confronted with antagonistic bacteria not sensitive to xenorhabdicin, form II cells were inferior to form I cells for providing protection for their nematode host (Sicard et al., 2005). The production of antibiotics is a large part of metabolism and comes at great cost. Without this cost, bacteria can invest more into nutrient uptake and proliferation and therefore better adapt to conditions outside of the tripartite relationship (Smigielski et al., 1994; Sicard et al., 2005).

The mechanisms for phenotypic change are beginning to be understood. In P. luminescens HexA negatively regulates primary form characteristics (Joyce & Clarke, 2003) while in X. nematophila Lrp positively regulates primary form characteristics (Cowles et al.,

2007), and for both bacteria the regulators affect both mutualism and pathogenesis.

Occasionally, reversion from form II to form I occurs in Xenorhabdus but has not been documented in Photorhabdus (Givaudan et al., 1995; Forst & Clarke, 2002). For detailed information about form-specific traits see Forst et al. (1997) and Forst and Clarke (2002).

Taxonomy and Systematics of Xenorhabdus and Photorhabdus

The current definition of species for bacteria is arbitrary and artificial but is universally operational (Stackebrandt et al., 2002). A species is described as a genomically coherent group of individual isolates that share a high degree of similarity in many independent features, and is diagnosable by a discriminative phenotype (Rosselló-Mora & Amann, 2001; Stackebrandt et al.,

2002; Adams et al., 2006). DNA:DNA homology remains the main criterion for the delineation of bacterial species. Strains within a species are recommended to have a DNA:DNA relatedness

o value of 70% or higher and a ∆Tm of 5 C or less (Wayne et al., 1987; Rosselló-Mora & Amann,

2001; Stackebrandt et al., 2002). However, the values should not be absolute for the description of a new species (Rosselló-Mora & Amann, 2001) and other molecular methods are encouraged as long as there is a sufficient degree of congruence with DNA:DNA reassociation (Stackebrandt et al., 2002). Based on the differences between 16S rRNA gene sequences and the low

DNA:DNA relatedness values of previous studies (Boemare et al., 1993; Nishimura et al., 1994),

Xenorhabdus species have been described without the incorporation of DNA:DNA reassociation

(Lengyel et al. 2005; Somvanshi et al., 2006). Together with the comparison of 16S rRNA sequences, Tailliez et al. (2006) included molecular typing profiles combining randomly amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) sequences, a reliable alternative to DNA:DNA hybridization. Profiles derived from ERIC sequences concatenated with REP (repetitive extragenic palindromic) and Box1A (BOX element) sequences have been used successfully to assess the diversity of Xenorhabdus and

Photorhabdus (Smith et al., 2003). This method should also be considered as it has been shown with other bacteria to have a strong congruence with DNA:DNA reassociation (Rademaker et al.,

2000).

A highly discriminative method of great promise for delineation of species is multi-locus sequence typing (MLST). MLST uses partial sequences of internal fragments from multiple housekeeping genes subjected to stabilizing selection, and the evolutionary distance between isolates is quantified based on the number of different loci (Maiden et al., 1998; Adams et al.,

2006). The method is highly reproducible and databases containing sequences and software are

publicly accessible for use in comparing the isolates of interest (see http://www.mlst.net and http://pubmlst.org). MLSTs have allowed for genomic affiliation of strains with much more confidence than by DNA:DNA reassociation (Lan & Reeves, 2001; Adams et al., 2006).

Although DNA similarity or congruent molecular methodology persists as the main criterion for delineation of bacterial species, a polyphasic approach for species description is the preferred method. Species descriptions should include an almost complete 16S rDNA sequence

(> 1300 nucleotides and < 0.5% ambiguity), the mol % G + C content of the type strain of the type genus (has been established for Xenorhabdus and Photorhabdus) and phenotype, including chemotaxonomic characters (Stackebrandt et al., 2002). Phenotypic data can be quickly and easily obtained by using standardized systems. API® substrate panels (BioMérieux, Inc.; http://www.biomerieux.com) for carbohydrate metabolism and other biochemical tests have been used in all the descriptions of Xenorhabdus and Photorhabdus species. In recent studies, Biolog

GN™ (Biolog, www.biolog.com), another standardized method of substrate utilization, has been employed for species descriptions and bacterial identification (Hazir et al., 2004; Lengyel et al.,

2005; Gouge & Snyder, 2006; Somvanshi et al., 2006). Phenotypes described by metabolism are useful but may be insufficient; therefore, the descriptions of other chemotaxonomic characters should be considered (Rosselló-Mora & Amann, 2001). Specific fatty acids can provide important taxonomic information (Dickstein et al., 2001) and fatty acid methyl ester profiles have been helpful for symbiont characterization (Janse & Smits, 1990; Suzuki et al., 1990;

Aguillera et al., 1993). Taxonomic characterization of bacteria can be done by using automated techniques (http://plantpath.ifas.ufl.edu/Fame/) and the data obtained from the strain(s) of interest are compared to databases containing information from other organisms.

For Xenorhabdus and Photorhabdus substantial differences in biochemical reactions of the same species have been reported in the literature (Holt et al., 1994; Brenner & Farmer, 2005), which may lead to difficulty in comparisons of species. Such variation is most likely a result of the use of different bacterial strains and/or phenotypic variants (Akhurst & Boemare, 1988).

Other possible factors for these differences may include weak and slow reactions and the media type used for biochemical characterization (Holt et al., 1994).

Xenorhabdus and Photorhabdus Taxonomy

The first symbiont of entomopathogenic nematodes was isolated from the DD136 strain of S. carpocapsae and described as a new bacterial species, Achromobacter nematophilus Poinar

& Thomas, 1965, by Poinar and Thomas (1965). The genus Achromobacter was later rejected

(Hendrie et al., 1974) and the described species were transferred to other genera. Since A. nematophilus did not fit into any of the accepted genera, the authors eventually erected a new genus, Xenorhabdus, to accommodate X. nematophilus and X. luminescens, symbionts of

Steinernema species and Heterorhabditis species, respectively (Thomas & Poinar, 1979).

Xenorhabdus luminescens was unquestionably distinct from other Xenorhabdus strains by both phenotypic (Akhurst, 1983a; Akhurst & Boemare, 1988; Boemare & Akhurst, 1988) and molecular characters (Grimont et al., 1984; Farmer et al., 1989; Suzuki et al., 1990). By using both the s1 nuclease and hydroxyapatite methods for determination of DNA:DNA similarity,

Grimont et al. (1984) separated X. luminescens isolates into a DNA relatedness group distinct from the other Xenorhabdus isolates. These findings were supported by the DNA relatedness studies conducted by Boemare et al. (1993). The lack of DNA homology to other Xenorhabdus species supported the proposal of Photorhabdus as a new genus for the symbionts of

Heterorhabditis (Boemare et al., 1993). Photorhabdus, although comparatively species poor, was found to be more homogenous than the species rich Xenorhabdus (Akhurst et al., 1996).

Currently, 20 species of Xenorhabdus and three species of Photorhabdus are recognized; however, the bacterial symbionts from the majority of recently isolated entomopathogenic nematode species have yet to be described.

Xenorhabdus and Photorhabdus are assigned to the family Enterobacteriaceae (Rahn,

1937) Ewing, Farmer & Brenner, 1980, within the gamma subdivision of the Proteobacteria.

Members of this family are Gram-negative rods, motile by peritrichous flagella or nonmotile, facultatively anaerobic, negative for oxidase, asporogenous, nonacid fast, chemoorganic heterotrophs with respiratory and fermentative metabolisms (Brenner, 1999; Brenner & Farmer,

2005). Xenorhabdus and Photorhabdus are considered phenotypically atypical to other members of this family (Holt et al., 1994). Xenorhabdus isolates are negative for the production of catalase, similar only to Shigella dysenteriae O group 1. Most Xenorhabdus and Photorhabdus are negative for the reduction of nitrate to nitrite, a trait shared only by some strains of Erwinia and Yersinia (Brenner, 1999; Boemare, 2002a; Brenner & Farmer, 2005). The DNA from species within most genera of Enterobacteriaceae is at least 20% homologous to each other and to Escherichia coli, the type species of the type genus of the Enterobacteriaceae; however,

Xenorhabdus and Photorhabdus are among the few notable exceptions (Brenner & Farmer,

2005).

Principal differences between Xenorhabdus and Photorhabdus are that Photorhabdus isolates are catalase positive and most are bioluminescent while all Xenorhabdus isolates are negative for both traits (Forst et al., 1997). In addition, Photorhabdus is distinguished from

Xenorhabdus by the occurrence of signature sequences in the 16S small subunit rDNA. The sequence TTCG is at positions 208-211 (E. coli numbering) in Xenorhabdus 16SrDNA, while

Photorhabdus has the longer TGAAAG at the same location (Szállás et al., 1997).

The polymorphism of the genes coding for the ribosomal RNA small subunit allows for assessment of bacterial diversity and/or the rapid identification of a bacterium in question so as to avoid laborious phenotypic characterization. Restriction fragment analysis of PCR amplified gene products is a method that has been used successfully for these purposes. Xenorhabdus and

Photorhabdus can be quickly and accurately identified on the basis of restriction fragment length polymorphisms of the 16S rRNA gene sequence (Brunel et al., 1997; Fischer-Le Saux et al.,

1998; Bonifassi et al., 1999) and placed into genotypes that correspond to DNA relatedness groups (Fischer-Le Saux et al., 1998). Phylogeny based on 16S rRNA sequences is also practical for the identification of Xenorhabdus and Photorhabdus strains (Liu et al., 2001;

Sergeant et al., 2006).

The Genus Xenorhabdus Thomas & Poinar, 1979

Xenorhabdus nematophila (= X. nematophilus) was divided into four subspecies based on variation in host nematode(s), pigmentation, maximum growth temperature, adsorption of dyes and other phenotypic characters (Akhurst, 1983a; Thomas & Poinar, 1983; Akhurst, 1986).

After a comprehensive phenotypic study (Boemare & Akhurst, 1988), the subspecies were elevated to the species X. nematophila, X. beddingii (Akhurst, 1986) Akhurst & Boemare, 1993,

X. bovienii (Akhurst, 1983) Akhurst & Boemare, 1993 and X. poinarii (Akhurst, 1983) Akhurst

& Boemare, 1993. Genomic DNA was hybridized and bacterial isolates coalesced into different

DNA relatedness groups that validated the described species (Boemare et al., 1993). A fifth species, X. japonica Nishimura, Hagiwara, Suzuki & Yamanaka, 1995 (originally described as X. japonicus) was proposed by Nishimura et al. (1994) on the basis of both phenotypic characterization and DNA:DNA homology. The bacterial nomenclature of Xenorhabdus was later revised by feminizing the species name to correspond to the feminine ending -rhabdus (i.e.,

X. nematophila and X. japonica) (Euzéby & Boemare, 2000). With the addition of the recently

described species X. budapestensis Lengyel, Lang, Fodor, Szállás, Schumann & Stackebrandt,

2005; X. cabanillasii Tailliez, Pagès, Ginibre & Boemare, 2006; X. doucetiae Tailliez, Pagès,

Ginibre & Boemare, 2006; X. ehlersii Lengyel, Lang, Fodor, Szállás, Schumann & Stackebrandt,

2005; X. griffiniae Tailliez, Pagès, Ginibre & Boemare, 2006; X. hominickii Tailliez, Pagès,

Ginibre & Boemare, 2006; X. indica Somvanshi, Lang, Ganguly, Swiderski, Saxena &

Stackebrandt, 2006; X. innexi Lengyel, Lang, Fodor, Szállás, Schumann & Stackebrandt, 2005;

X. koppenhoeferi Tailliez, Pagès, Ginibre & Boemare, 2006; X. kozodoii Tailliez, Pagès, Ginibre

& Boemare, 2006; X. mauleonii Tailliez, Pagès, Ginibre & Boemare, 2006; X. miraniensis

Tailliez, Pagès, Ginibre & Boemare, 2006; X. romanii Tailliez, Pagès, Ginibre & Boemare, 2006;

X. stockiae Tailliez, Pagès, Ginibre & Boemare, 2006 and X. szentirmaii Lengyel, Lang, Fodor,

Szállás, Schumann & Stackebrandt, 2005) (Hazir et al., 2004; Lengyel et al., 2005; Tailliez et al., 2006), some 20 species have been proposed in the genus.

Characters of Xenorhabdus

Xenorhabdus are asporogenous, rod-shaped Gram-negative cells measuring 0.3-2 µm by

2-10 µm and with occasional filaments that are 15-20 µm long (Akhurst & Boemare, 2005).

Spheroplasts that average 2.6 µm in diam. occur at the end of exponential growth (Akhurst &

Boemare, 2005). Cells occur in two forms. They are facultatively anaerobic with both respiratory and fermentative types of metabolism, are negative for oxidase and catalase and generally do not reduce nitrate to nitrite (Akhurst & Boemare, 2005; Brenner & Farmer, 2005).

Optimum growth temperature is 28C or less, with some strains able to grow at temperatures as high as 42C (Akhurst & Boemare, 2005; Tailliez et al., 2006). Acid but not gas is produced from glucose. N-acetylglucosamine, glycerol and mannose are fermented and most species ferment fructose (Akhurst & Boemare, 2005; Lengyel et al., 2005). Form I cells are motile by

peritrichous flagella (Givaudan et al., 1995). Cells generally produce protoplasmic crystalline inclusions during the stationary phase of growth (Akhurst & Boemare, 2005). Most strains are lipolytic and positive for DNase and protease (Boemare & Akhurst, 1988). The G + C mol% of the DNA is 43-50 as measured by DNA equilibrium buoyant density. The bacteria associate with nematodes of the family Steinernematidae. The type species is X. nematophila.

The Genus Photorhabdus Boemare, Akhurst & Mourant, 1993

The species X. luminescens was erected to accommodate the luminescent symbiotic bacteria isolated from the genus Heterorhabditis (Thomas & Poinar, 1979). Strains within this species were found to be phenotypically (Akhurst, 1983a) and genotypically (Grimont et al.,

1984) divergent from the symbionts of steinernematid nematodes. Grimont et al. (1984) demonstrated that X. luminescens isolates formed two DNA relatedness groups that were dissimilar to the DNA relatedness groups of X. nematophila and X. bovienii. Clinical isolates of

X. luminescens revealed a fifth DNA hybridization group, forming three DNA relatedness groups within X. luminescens (Farmer et al., 1989). Today we know that these groups correspond to the species P. luminescens, P. temperata Fischer-Le Saux, Viallard, Brunel, Normand & Boemare,

1999 and P. asymbiotica. Chemotaxonomic characteristics and DNA relatedness data (Akhurst

& Boemare, 1988; Boemare & Akhurst, 1988; Suzuki et al., 1990) strongly supported the transfer of X. luminescens to a new genus and, accordingly, Photorhabdus was proposed by

Boemare et al. (1993). Further study confirmed that Photorhabdus was clearly divided into two distinct groups: i) nematode symbionts; and ii) clinical specimens (Akhurst et al., 1996). The symbiotic group appeared to be fairly heterogenous, and phylogenetic analysis of 16S rRNA gene sequences supported by DNA relatedness provided evidence for heterogeneity within this group (Szállás et al., 1997). The combined information derived from phenotypic characterization, DNA relatedness and phylogenetic inference based on 16S rDNA, supported

the proposal of three subspecies of P. luminescens, including P. luminescens subsp. luminescens

(Thomas & Poinar, 1979) Boemare, Akhurst & Mourant, 1993, P. luminescens subsp. akhurstii

Fischer-Le Saux, Viallard, Brunel, Normand & Boemare, 1999, and P. luminescens subsp. laumondii Fischer-Le Saux, Viallard, Brunel, Normand & Boemare, 1999, plus two additional species, P. temperata and the clinical strain, P. asymbiotica (Fischer-Le Saux et al., 1999b).

Peel et al. (1999) isolated P. asymbiotica from human specimens in Australia which, according to PCR-RFLP analysis of 16S rDNA, formed a homogeneous group that differed from the described American isolates. Photorhabdus asymbiotica subsp. australis Fischer-Le Saux,

Viallard, Brunel, Normand & Boemare, 1999 was proposed to include these isolates (Akhurst et al., 2004). Additionally, two new subspecies of P. luminescens have been described; P. luminescens subsp. kayaii Hazir, Stackebrandt, Lang, Schumann, Ehlers & Keskin, 2004 and P. luminescens subsp. thracensis Hazir, Stackebrandt, Lang, Schumann, Ehlers & Keskin, 2004

(Hazir et al., 2004). Currently, three species of Photorhabdus are recognized.

Characters of Photorhabdus

Photorhabdus are asporogenous, rod-shaped, Gram-negative cells measuring 0.5-2  1-

10 µm with occasional filaments up to 30 µm in length (Boemare & Akhurst, 2005). The cell size is highly variable. Spheroplasts that average 2.6 µm in diam. occur at the end of exponential growth and during stationary growth (Boemare & Akhurst, 2005). Cells occur as two phenotypic variants. Optimum growth temperature is 28C and some strains are able to grow at 40C

(Akhurst et al., 2004). On nutrient agar, most strains produce pink, red, orange, yellow, or green pigment (Boemare & Akhurst, 2005). Most strains are bioluminescent, which is usually detectable by the dark-adapted human eye. They are facultatively anaerobic, chemoorganotrophs with both respiratory and fermentative types of metabolism, negative for oxidase,

positive for catalase, and do not reduce nitrate to nitrite (Brenner & Farmer, 2005). A discriminating factor for Photorhabdus is hemolytic patterns on blood agar (see next paragraph).

Most strains are hemolytic for sheep and/or horse blood agars. Photorhabdus produce protease as determined by the lysis of gelatin. Cells are lipolytic and able to hydrolyze Tween 20 with many strains able to hydrolyze Tweens 40, 60, 80 and 85. Acid, but not gas, is produced from glucose, N-acetylglucosamine, fructose, mannose, maltose and ribose, but weakly so from glycerol (Boemare & Akhurst, 2005; Akhurst et al., 2004; Hazir et al., 2004). Fumarate, glucosamine, L-glutamate, L-malate, L-proline, succinate and L-tyrosine are assimilated. Cells are motile by peritrichous flagella. Most cells produce protoplasmic crystalline inclusions during the stationary phase of growth (Boemare & Akhurst, 2005). The G + C mol% of the DNA is 43-

45. The type species is P. luminescens.

Farmer et al. (1989) described an unusual hemolytic reaction on sheep blood agar by

Photorhabdus in which there was no hemolysis immediately surrounding the colony. Instead hemolysis occurs as a thin line (2 mm) 6-13 mm away from the colony edge (Farmer et al., 1989;

Peel et al., 1999). It was referred to as “Xenorhabdus luminescens hemolysis” because it was thought to be unique to this group of bacteria. This phenotypic character is not observed in

Xenorhabdus naturally and accordingly, Akhurst et al. (1996) proposed the new term “annular hemolysis” because of the formation of an annulus that encircles each colony. It should be noted that this type of hemolysis is not exclusive to Photorhabdus but also occurs in Bacillus cereus for which it is identified as a “discontinuous hemolytic pattern” (Beecher & Wong, 1994).

Annular hemolysis, a descriptive character of most isolates of P. asymbiotica (Weissfeld et al.,

2005), does not occur in all strains of Photorhabdus and only rarely in P. luminescens subsp. laumondii (Fischer-Le Saux et al., 1999b; Peel et al., 1999; Akhurst et al., 2004).

Nematode/Bacteria Associations

Each entomopathogenic nematode is associated with a specific bacterial symbiont. In nature, the nematode develops and reproduces in a monoxenic environment that is established by its symbiont within an insect cadaver. The relationships between entomopathogenic nematodes and their symbiotic bacteria are summarized in Table 1. Each species of Steinernema is mutualistically associated with only one species of Xenorhabdus; however, some species of

Xenorhabdus are symbionts of multiple steinernematid species. Symbionts with multiple host associations include: Xenorhabdus beddingii, the symbiont of S. longicaudum and an undescribed nematode species; X. bovienii, the symbiont of S. affine, S. feltiae, S. intermedium and S. kraussei; X. hominickii, the symbiont of S. karii and S. monticolum, X. kozodoii, the symbiont of S. arenarium and S. apuliae and X. poinarii, the symbiont of S. glaseri and S. cubanum (Akhurst & Boemare, 1988; Fischer-Le Saux et al., 1999a; Boemare, 2002a, b). In contrast to the steinernematid-Xenorhabdus complex, different strains of the nematode species

H. bacteriophora are associated with two different species of Photorhabdus and subspecies thereof. The Brecon and HP88 groups are associated with the subspecies P. luminescens subspecies luminescens and P. luminescens subspecies laumondii, respectively, whereas the NC group is associated with P. temperata (Fischer-Le Saux et al., 1999b). Photorhabdus temperata is also associated with H. downesi, H. megidis and H. zealandica. Other multiple symbiotic associations may exist but are not yet known for the symbiotic bacteria of the most recently described nematode species have yet to be characterized.

Gnotobiological techniques have been used to investigate the level of specificity between the nematode host and its symbiont. Nematodes are freed from their symbiont and then associated with a bacterium of interest, either the natural symbiont or another potential partner.

It is possible to rear Steinernema axenically, but an artificial diet has not yet been developed for

Heterorhabditis (Boemare et al., 1997b). However, bacteria-free infective juveniles of H. bacteriophora and H. indica can be produced by culturing the nematodes on P. temperata and the symbiont of H. bacteriophora H06 respectively (Han & Ehlers, 1998). Otherwise, surface- sterilized eggs are harvested from Heterorhabditis and combined immediately with the symbiont

(Boemare et al., 1997b). Due to the specificity of the host-symbiont relationship, heteroxenic associations are difficult to establish (Boemare et al, 1997b; Bonifassi et al, 1999). Specificity is determined on three levels: recovery of the IJ, nutritive properties to support nematode reproduction and development, and retention of the bacteria in the intestinal tract of the IJ

(Grewal et al, 1997; Han & Ehlers, 1998).

Grewal et al.(1997) reported that a food signal was secreted in the cell-free filtrate of the

S. scapterisci symbiont. Although, S. scapterisci is able to use and retain symbionts of other steinernematids, the recovery was delayed but improved upon supplementation with the cell-free filtrate of its natural symbiont (Grewal et al., 1997). This indicated a level of specificity between the nematode and its symbiont for IJ recovery.

Strauch and Ehlers (1998) also reported that an unknown food signal is secreted in the medium by the bacterial symbiont. It is required for the recovery of Heteorhabditis from its quiescent IJ stage to continue its parasitic life cycle (Strauch & Ehlers, 1998). The food signal appears to be a universal signal recognizable by H. bacteriophora since upwards of 50% of IJ are able to recover in response to both P. luminescens akhurstii and P. temperata (Han & Ehlers,

1998). Han and Ehlers (1998) established that the signals responsible for nematode recovery are not the same compounds necessary for completion of the life cycle. P. luminescens bacteria isolated from H. indica are nutritionally incompatible for H. bacteriophora H06, and the nematodes are unable to develop (Han & Ehlers, 1998). However, Gerritsen et al. (1998)

produced a successful monoxenic combination of H. megidis with P. luminescens. The nematodes could use an alternative symbiont, but virulence was reduced (Gerritsen et al., 1998).

The best nutritional condition for nematode growth is not necessarily established by its indigenous symbiont, but nematode growth appears to be unrelated to nematode virulence (Han et al., 1991).

Some combinations of nematodes with nonsymbionts support nematode development; however, the nonsymbiont does not maintain long-term associations (Akhurst & Boemare, 1990;

Ehlers et al., 1990; Han & Ehlers, 1998). Other bacteria may produce conditional associations with a nematode, but the symbiont is the most efficient partner for pathogenicity, reproduction, and development of the nematode (Bonifassi et al., 1999).

Some combinations of Steinernema spp. with non-native Xenorhabdus spp. support in vitro nematode growth and development (Akhurst, 1983b; Grewal et al., 1997; Sicard et al.,

2004b). However, Sicard et al. (2004b) found that, within an insect host, non-native

Xenorhabdus spp. other than X. poinarii were pathogenic to S. carpocapsae, leading to the assumption that a host nematode may have evolved to be resistant to virulence factors produced by its own symbiotic bacteria. This selective resistance could be an advantage given to the nematode by their symbiotic bacteria when confronted with coinfection of an insect by another nematode species (Sicard et al., 2004b). Other bacteria may produce conditional associations with a nematode, but the natural symbiont is the most efficient partner for pathogenicity, reproduction and development of the nematode (Bonifassi et al., 1999; Sicard et al., 2004b).

The specificity between the nematode and its bacterial symbiont is a result of the exclusion of bacterial competitors and a specific recruitment by the IJ of its symbiont. Both

Xenorhabdus and Photorhabdus produce bacteriocins, which are proteinaceous compounds with

antimicrobial activity against closely related bacterial strains or species. The bacteriocins are functionally important for the symbiotic relationship as they help the natural symbiont to out compete closely related bacteria (Boemare et al., 1997a; Sicard et al., 2005). Xenorhabdicin is a phage tail-like bacteriocin isolated from X. nematophila that has antibiotic activity against other

Xenorhabdus spp., P. luminescens and species of Proteus (Boemare et al., 1992; Thaler et al.,

1995). Lumicins are bacteriocins isolated from P. luminescens and are active against other

Photorhabdus spp. as well as distantly related bacteria such as E. coli (Sharma et al., 2002). The latter suggests that these bacteriocins may not be exclusively active against competing symbionts but may be involved in clearing of the insect gut microflora (Sharma et al., 2002).

For the symbiotic relationship to persist, the bacteria must be able to colonize their host nematode. Fimbriae are bacterial cell surface appendages and have been proposed to be involved in host colonization, specifically for association with the epithelial cells of the nematode gut (Binnington & Brooks, 1993; Moureaux et al., 1995; Forst & Nealson, 1996); however, the role of the fimbriae has not yet been ascertained (Ciche et al., 2006). Xenorhabdus nematophila produces mannose-resistant fimbriae, resembling the fimbriae of Proteus mirabilis

(Moureaux et al., 1995; Forst & Clarke, 2002). The genome of P. luminescens subsp. laumondii strain TT01 was sequenced and was found to have a large repertoire of fimbrial genes (Duchaud et al., 2003). Among the fimbrial genes were two gene clusters encoding proteins similar to the mannose-resistant fimbriae and on another gene cluster, ngrA, which in Photorhabdus is important for bacteria-nematode interactions (Ciche et al., 2001; Duchaud et al., 2003). These genes too may play a role in nematode colonization (Duchaud et al., 2003).

Several genes involved in vesicle colonization of the host nematode have been identified in X. nematophila. Among these is a novel class of genes, designated nil for nematode intestine

localization, which may have a specific role for allowing the bacterium to colonize the nematode host (Heungens et al., 2002). A small transmembrane protein of unknown function is encoded by nilA; nilB encodes an outer membrane protein and nilC encodes an outer membrane associated lipoprotein (Heungens et al., 2002; Cowles & Goodrich-Blair, 2004). nilA is in part required for vesicle colonization whereas the gene products for nilB and nilC are essential

(Heungens et al., 2002; Cowles & Goodrich-Blair, 2004). Also, cells may be colonization- defective due to mutations in the regulatory proteins RpoS, RpoE and Lrp (Vivas & Goodrich-

Blair, 2001; Heungens et al., 2002).

In E. coli, the transcription factor, RpoS, controls regulons that can mediate stress resistance and survival. In X. nematophila, RpoS is essential for nematode colonization and helps to mediate resistance to peroxide stress. However, to date, the genes regulated by RpoS in

X. nematophila are unknown (Vivas & Goodrich-Blair, 2001). Very few cells with a defective extracytoplasmic stress sigma factor, σE, are able to colonize the vesicle, and this defect may be related to the reduced survival of rpoE mutants during the colonization assay (Heungens et al.,

2002).

While RpoS and σE are not necessary for expression of the nil genes, they may regulate other factors involved in colonization to promote survival within the intestinal vesicle (Vivas &

Goodrich-Blair, 2001; Heungens et al., 2002). The regulatory protein Lrp is required, synergistically with a small transcription factor NilR, to repress the expression of nilA, B, and C, and cells defective in either the nil genes or lrp lose the ability to colonize the host (Cowles &

Goodrich-Blair, 2004, 2006; Cowles et al., 2007). The nil loci are not present in the P. luminescens subsp. laumondii genome (Ciche et al., 2006); however, the pbgPE operon, which is required for pathogenicity, is also required by P. luminescens for colonization of the IJ intestine

(Bennett & Clarke, 2005). Our understanding of the nematode-bacterium symbiosis is growing but requires further study to explain the provisions given by the symbiont and the resilience of the association over many generations (Bonifassi et al., 1999; Boemare, 2002b).

Though the entomopathogenic nematodes share an exclusive mutualistic relationship with their respective symbiotic bacteria, other bacteria are occasionally isolated from the nematode or the host insect subsequent to infection. Some of these bacteria include members of the genera Acinetobacter, Citrobacter, Ochrobactrum, Providencia, Pseudomonas, Serratia,

Salmonella and Vibrio, and are likely associated with the cuticle or found between the L2 and L3 layers of the nematode cuticle (Lysenko & Weiser, 1974; Aguillera & Smart, 1993; Jackson et al., 1995; Bonifassi et al., 1999; Babic et al., 2000; Walsh & Webster, 2003; Gouge & Snyder,

2006). Some bacteria are able to persist alongside the symbiont despite the virulence factors produced by the symbiotic bacteria. Acinetobacter, a bacterium carried by the nematode, is able to endure the antimicrobial compounds produced by X. bovienii and proliferate inside the host insect cadaver (Walsh & Webster, 2003). Ochrobactrum spp. were found to associate naturally with P. luminescens subsp. akhurstii, the symbiont of H. indica (Babic et al., 2000) and

Ochrobactrum anthropi has been isolated with the symbiont of H. floridensis (Koppenhöfer &

Jones, data unpubl.). Paenibacillus nematophilus has a stable relationship with Heterorhabditis spp. The bacterial sporangia adhere to the cuticle of the IJ, are resistant to the Photorhabdus virulence factors and reproduce in sympatry in the insect host (Enright et al., 2003; Enright &

Griffin, 2004, 2005). It is possible for nematodes to transport other nonsymbiotic bacteria and these dixenic associations may be detrimental to the nematode (Enright & Griffin, 2005) or the symbiotic relationship (Bonifassi, 1999; Walsh & Webster, 2003).

Phylogenetic Systematics

Evolutionary relationships of members of the family Enterobacteriaceae derived from small subunit RNA are shown in Figure 1 (Francino et al., 2003). The phylogenetic tree was constructed using 16S rDNA sequences and rooted with Vibrio cholerae, a member of the family

Vibrionaceae, also part of the gamma subclass of Proteobacteria. Phylogenetic analyses reveal that within Enterobacteriaceae, Xenorhabdus and Photorhabdus form a monophyly (Szállás et al., 1997) with Proteus as their closest relative (Forst et al., 1997; Szállás et al., 1997; Francino et al., 2003). This is in agreement with the phenotypic traits of Xenorhabdus and Photorhabdus.

Both symbionts are atypical of the genera belonging to Enterobacteriaceae (Holt et al., 1994), being similar only to each other. Branch lengths between X. nematophila and P. asymbiotica are long in comparison to those of other genera included in the phylogeny and this too is supported by chemotaxonomic characterization.

Phylogenies investigating the inter- and intrageneric relationships of Xenorhabdus and

Photorhabdus have been constructed using 16S rRNA gene sequences (Rainey et al., 1995;

Suzuki et al., 1996; Liu et al., 1997, 2001; Szállás et al., 1997; Fischer-Le Saux et al., 1999b;

Marokhazi et al., 2003; Akhurst et al., 2004; Hazir et al., 2004; Lengyel et al., 2005; Sicard et al., 2005; Sergeant et al., 2006; Tailiez et al., 2006). Relationships of Xenorhabdus species are shown in the phylogenetic tree in Figure 2 modified after Tailliez et al., 2006. This is a NJ tree constructed using nearly complete 16SrDNA sequences and rooted with P. luminescens subsp. laumondii (Tailliez et al., 2006). Species of Xenorhabdus coalesced into 13 groups with strong bootstrap support (Tailliez et al., 2006). However; not all of the branch nodes have high support values (e.g., the clades with the symbionts of S. oregonense and S. monticolum), indicating that branching order is not necessarily resolved. Phylogenetic relationships among presently recognized species of Photorhabdus are presented in Figure 3 (Akhurst et al., 2004). In this

scenario, Photorhabdus diverges into two clades that are inconsistent with species-level groupings (Akhurst et al., 2004). Photorhabdus asymbiotica subsp. asymbiotica diverges from the other Photorhabdus as sister taxon to P. luminescens subsp. luminescens, while P. asymbiotica subsp. australis forms a monophyletic group with other P. luminescens subspecies.

The branch support values are weak or nonexistent at nodes that join interspecific taxa. The phylogeny based on small subunit rRNA gene sequences supports the subspecies-level groupings of Fischer-Le Saux et al. (1999b) but is not suitable for distinguishing species within

Photorhabdus (Akhurst et al., 2004).

The use of 16S rRNA gene sequences for determining phylogenetic relationships has limitations. This is a variable that should be considered for interpretation of evolutionary history between taxa. Like other bacterial genes, ribosomal genes can undergo lateral gene transfer across diverse taxonomic groups or be recombined, which could lead to artificial evolutionary data (Yap et al., 1999). Consequently, resolving bacterial phylogeny based solely on 16S rRNA gene sequences may not be sufficient (Lerat et al., 2003). Akhurst et al. (2004) were able to demonstrate that a phylogeny based on the gyrB gene provided a more robust tree for determining intrageneric relationships among Photorhabdus. This is due to the lower proportion of invariant nucleotide positions than in the 16S rRNA gene (Akhurst et al., 2004). Figure 4 is a

NJ tree based on the partial gyrB sequences of Akhurst et al. (2004). At deeper nodes, the taxa are divided by species, then at the branches near the terminus, the taxa are subdivided by subspecies. Unlike in the 16S rRNA gene-based phylogeny, P. asymbiotica subsp. asymbiotica and P. asymbiotica subsp. australis form a monophyletic group that diverges as sister taxon to P. luminescens. The subspecies of P. luminescens also form a monophyletic group. This is further supported by the Photorhabdus phylogeny based on concatenated sequences of glnA and gyrB

constructed by Gerrard et al. (2006). Recently, Sergeant et al. (2006) characterized

Xenorhabdus isolates by multi locus sequence typing in which a portion of four housekeeping genes were sequenced, concatenated and analyzed. When compared to analysis by 16S rRNA sequences, the sequence typing was more informative and able to differentiate between strains that were undistinguishable by 16S rDNA sequence data alone (Sergeant et al., 2006). Although

16S rRNA gene sequence data are informative and play an integral role for bacterial systematics, to resolve the intrageneric relationships for both Xenorhabdus and Photorhabdus more robust trees should be developed by comparing sequences from additional strains from carefully selected protein-coding genes.

Coevolution

Unique to the nematode/bacterium complexes is the involvement of three partners

(bacterium, nematode and insect) and the presence of both mutualistic and parasitic relationships.

The bacterium is an ecologically obligate partner (Boemare et al., 1997a) because it is unable to exist in the environment without the nematode host. In turn, the bacterium is required by the nematode to complete its life cycle efficiently (Forst & Nealson, 1996). In particular,

Heterorhabditis spp. are unable to develop in culture without Photorhabdus (Boemare et al.,

1997b). Furthermore, both nematode and bacterium are implicated in the parasitic relationship with the insect. Although the Xenorhabdus-Steinernema and Photorhabdus-Heterorhabditis symbioses are very similar in life cycle and pathogenicity, they differ in how the bacteria are released into the insect, colonization of the host nematode and the specificity of the relationship.

The bacteria differ from each other as the genome of Photorhabdus is about 25% larger than that of Xenorhabdus (Forst & Goodner, 2006) and the bacteria produce different virulence and symbiotic factors. According to Blaxter et al. (1998), bacteria harboring entomopathogenic nematodes evolved more than once, and therefore, it seems fair to assume that similarities

between these two systems have most likely resulted from convergent evolution (Boemare,

2002a).

Coevolution has been defined as the evolution of reciprocal adaptations in lineages that are ecologically associated (Page 2003), and in mutualistic relationships often both host and symbiont evolve to accommodate each other (Moran, 2006). Although still much is to be learned of the nematode/bacterium symbiosis, some examples of adaptations have been observed. For instance, steinernematid nematodes possess an intestinal vesicle specifically for housing their symbiotic bacteria (Bird & Akhurst, 1983). A similar structure is not found in

Heterorhabditis spp. However, both genera of bacteria express genes that are specific for colonization of the nematode host (Bennett & Clarke, 2005; Cowles & Goodrich-Blair, 2004,

2006; Cowles et al., 2007). Also, nematodes are able to withstand the virulence factors of their bacterial partners whereas the virulence factors of other bacterial symbionts may have deleterious effects (Sicard, 2004). Due to the tight nature of the nematode/bacterium association, it is plausible that their evolutionary history is shared. Phylogenetic analyses of molecular sequence data obtained from both host and symbiont are helpful in establishing congruence that may be occurring along these presumed parallel lineages (Nishiguchi, 2001). The nematode and bacteria phylogenies have yet to be compared.

This chapter was originally published as part of Koppenhöfer (2007). Since this publication, many advances have been made in the studies of Xenorhabdus and Photorhabdus.

The updated taxonomy and systematics of these bacteria are presented in the following chapters.

Aims and Objectives

To explore the genetic diversity among strains of Xenorhabdus and Photorhabdus, intrageneric relationships were analyzed by generating genomic fingerprints based on the amplification of repetitive DNA sequences distributed throughout the chromosome (rep-PCR).

Analyses of the resulting genomic fingerprints revealed the formation of distinct groups from which representative strains were selected for the construction of bacterial phylogenies.

Sequences from portions of housekeeping genes and the 16S rRNA gene were used to construct phylogenetic trees, and the molecular phylogenies inferred for each locus separately and for concatenated sequences were investigated.

The following objectives were then undertaken to answer basic questions with regard to relationships among Photorhabdus with a special emphasis on strains isolated from

Hetororhabditis in Florida and among strains of Xenorhabdus.

Objective 1: To determine the genetic diversity and phylogenetic relationships among Photorhabdus species and strains thereof isolated in Florida is presented in the second chapter. The relative phylogenetic utility of the RNA polymerase B gene (rpoB) was assessed and compared to that of DNA gyrase B (gyrB). Partial sequences derived from rpoB and gyrB were used separately and combined to infer phylogeny. The ensuing phylogenies were then compared to established Photorhabdus phylogenies (Akhurst et al., 2004; Gerrard et al., 2006; Tailliez et al., 2012; Ferreira et al., 2013b; 2014) with special regard to the position of the strains isolated from Florida.

Objective 2: To determine the genetic diversity and phylogenetic relationships among Xenorhabdus species isolated from multiple steinernematid nematode hosts were evaluated. Four housekeeping genes (gyrB, groEL, recA and rpoB) and the 16S rRNA gene were employed individually and pooled together to infer Xenorhabdus phylogeny. Evidence is presented here suggesting that the most robust phylogeny is constructed from the concatenated housekeeping gene sequences.

Table 1-1. Symbiotic Associations Xenorhabdus/Steinernemaa X. nematophilab S. carpocapsae, S. anatoliense, S. websteric X. beddingii S. longicaudum X. bovienii S. affine, S. feltiae, S. intermedium, S. jollieti, S. kraussei,S. oregonense, S. puntauvense, S. weiseric X. budapestensis S. bicornutum X. cabanillasii S. riobrave X. doucetiae S. diaprepesi X. ehlersii S. serratum (nomen nudum) X. griffiniae S. hermaphroditum X. hominickii S. karii, S. monticolum X. innexi S. scapterisci X. indica S. thermophilum (S. abbasi), S. yirgalemensed X. ishibashiie S. aciari X. japonica S. kushidai X. khoisanef S. khoisanae X. koppenhoeferi S. scarabaei X. kozodoii S. arenarium, S. apuliae, S. boemareic X. magdalensisg S. australe X. mauleonii S. sp. isolated from the island of St. Vincent located in the Caribbean Sea X. miraniensis Nematode of the family Steinernematidae isolated from Mirani, Queensland Australia. X. poinarii S. glaseri, S. cubanum X. romanii S. puertoricense X. stockiae S. siamkayai X. szentirmaii S. rarum, S. costaricensec X. vietnamensish S. sangi Photorhabdus/Heterorhabditisa P. luminescens subsp. luminescensb H. bacteriophora Brecon, H. floridensisi P. luminescens subsp. akhurstii H. indica, H. bacteriophoraj P. luminescens subsp. caribbeanensish H. bacteriophora, Heterorhabditis sp. P. luminescens subsp. hainanensish Heterorhabditis sp. P. luminescens subsp. kayaii H. bacteriophora Turkey P. luminescens subsp. kleiniik H. georgiana, H. bacteriophora P. luminescens subsp. laumondii H. bacteriophora HP88, H. safricanal P. luminescens subsp. noenieputensism H. noenieputensis P. heterorhabditisn H. zealandica P. temperata subsp. temperata H. megidis P. temperata subsp. cinerea H. downesi, H. megidis P. temperata subsp. khaniih H. bacteriophora, H. megidis, H. georgianaj P. temperata subsp. stackebrandtiio,h,m H. bacteriophora, H. megidis, H. georgiana Heterotypic synonym of P. t.ssp. khanii

Table 1-1. Continued Photorhabdus/Heterorhabditisa P. temperata subsp. tasmaniensish H. zealandica, H. marelatus P. temperata subsp. thracensisg H. bacteriophora, Turkey P. asymbiotica subsp. asymbiotica unknown host P. asymbiotica subsp. australis unknown host P. asymbitotica strain Kingscliff H. gerrardip aThis chapter excluding the “Aims and Objectives” was originally published as a part of Koppenhöfer, 2007. This table has been updated and reflects the current symbiotic associations. References that are listed here are not in text. bType species of Xenorhabdus and Photorhabdus c Lee & Stock, 2010a dFerreira et al., 2016 eKuwata et al., 2013 fFerreira et al., 2013a gTailliez et al., 2012 hTailliez et al., 2010 iShapiro-Ilan et al., 2014 jManeesakorn et al 2011 kAn &Grewal, 2011 lGeldenhuys et al., 2016 mFerreira et al., 2013b nFerreira et al., 2014 oAn & Grewal, 2010 pPlichta et al., 2009

Escherichia coli Salmonella enterica Erwinia amylovera Citrobacter freundii Enterobacter intermedius Serratia marcescens Yersinia enterocolitica Yersinia pestis Providencia stuartii Morganella marganii Proteus vulgaris Photorhabdus asymbiotica Xenorhabdus nematophila Edwardsiella tarda Bruchnera aphidicola strain Sg Vibrio cholerae

Figure 1-1. 16S rRNA gene-based phylogeny for relationships of genera within the family Enterobacteriaceae. To view tree in its entirety, see Francino et al. 2006.

78 Breton, DQ282116

77 75 DSM3370, AY278674 56 X. nematophila 100 CB6, AF522294 100 DSM 3370, X82251

100 SK72, AY521239 X. poinarii 100 DSM 4768, X82253

65 DSM 4766, X82252 66 X. bovienii 97 62 CB54, AY317154 85 79 100 2B, X. sp. (S. oregonense) 76 100 1A, X. sp. (S. oregonense)

CB43, DQ329379 63 100 X. budapestensis 79 85 100 DSM 16342, AJ810293 90 96 DSM 16337, AJ810294, X. ehlersii 92 DSM 16336, AJ810292 76 55 100 75 X. innexi 80 100 UY61, AY521243 86

88 2B, X. sp. (S. monitcolum)

SK-1 IAM14265, D78008, X. japonica 100

100 83 DSM 16338, AJ810295, X. szentirmaii 66 X. sp., 13 (S. sp.,Vietnam)

AY521244, X. sp., USTX62 (S. riobrave)

DSM 4764, AY278675, X. beddingii

Figure 1-2. Phylogenetic relationships of Xenorhabdus based on maximum likelihood analysis of partial 16S rDNA sequences (Koppenhöfer, 2007). Bootstrap support indices (1000 replications) for neighbor joining (above branch) and maximum parsimony (below branch) analyses are indicated where congruent with maximum likelihood tree. Species of Photorhabdus chosen for outgroup. Tree rooted with P. luminescens ssp. akhurstii (AJ007359), P. temperata (AY278655) and P. asymbiotica ssp. asymbiotica (AY278672). TrN+I+G model of evolution (γ shape parameter = 0.5621; substitution rate matrix Ra = 1.0000, Rb = 2.8789, Rc = 1.0000, Rd = 1.0000, Re = 4.6016, Rf = 1.0000; base frequencies: freqA = 0.2459, freqC = 0. 2210, freqG = 0. 3271, freqT = 0.2060).

TT01, BX571873

72 TT01, BX571863 94 P. luminescens subsp. 60 77 HP88, AY278648 laumondii

53 Brecon, AY278647 90 1121, AJ560630 74 P. luminescens subsp. 100 100 47-10, AJ560630 kayaii

W14, AY278642 100 P. luminescens subsp. 99 FRG04, AJ007359 akhurstii

39-8, AJ560634, P. luminescens subsp. thracensis

AY278670, P. sp. JUN 9802892, AY280572 100 P. asymbiotica subsp. 100 GCH001, AY280574 australis

100 ATCC 43952, Z76753 100 100 ATCC 43950, Z76755 P. asymbiotica subsp. 100 asymbiotica 53 ATCC 43951, Z76754 61 Hb, X82248 99 P. luminescens subsp. 100 Hm, AY278641 luminescens

AY216500, P. sp. Q614

K122, AY278651 99 93 AY296252 99 80 XlNach, AJ007405 80 P. temperata 62 HL81, AY278653

99 Meg1, AY278655 99 NC19, AY278657 Figure 1-3. Phylogenetic relationships of Photorhabdus based on maximum likelihood analysis of partial 16S rDNA sequences (Koppenhöfer, 2007). Bootstrap support indices (1000 replicates) for neighbor joining (above branch) and maximum parsimony (below branch) analyses are indicated where congruence resided with maximum likelihood tree. Tree rooted with Pr. vulgaris (X07652) and S. proteomaculans (AJ233435). TrN+I+G model of evolution (γ shape parameter = 0.6219; substitution rate matrix Ra = 1.0000, Rb = 2.9659, Rc = 1.0000, Rd = 1.0000, Re = 3.2786, Rf = 1.0000; base frequencies: freqA = 0. 2559, freqC = 0. 2173, freqG = 0. 3181, freqT = 0. 2087).

D1 AY278499 100 Tetuan 100 100 AY278515 P. luminescens subsp. akhurstii 62 63 K81 AY278510 61 Hb 100 AY278501 100 P. luminescens subsp. luminescens 100 Hm 100 AY278505 TT01 100 BX571859 99 100 HP88 P. luminescens subsp. laumondii 81 AY278508 P. sp. Q614 AY278514 100 GCH001 100 AY278500 100 100 9802892 P. asymbiotica subsp. australis 54 AY278496 100 60 1216-79 100 AY278492 100 P. asymbiotica subsp. asymbiotica 100 3265-86 AY278494 NZH3 AY278513 Meg 99 AY278512 100 C1 AY278497 P. temperata 83 XlNach 100 AY278517 100 HL81 AY278504 Serratia proteomaculans AJ300531 Escherichia coli X04341

Figure 1-4. Phylogenetic relationships between species/strains of Photorhabdus based on Maximum likelihood analysis of partial gyrB gene sequences (Koppenhöfer, 2007). Bootstrap support indices (1000 replications) for Neighbor Joining and Maximum Parsimony analyses are indicated above and below the branches, respectively where congruent with maximum likelihood tree. TrNef+I+G model of evolution by hLRT (γ shape parameter = 0. 9677; substitution rate matrix Ra = 1.0000, Rb = 4.6683, Rc = 1.0000, Rd = 1.0000, Re =, Rf = 1.0000; equal base frequencies).

CHAPTER 2 DIVERSITY OF Photorhabdus SPP. ISOLATED IN FLORIDA

Introduction

Entomopathogenic nematodes of the family Heterorhabditidae are characterized by a mutualistic association with bacteria of the genus Photorhabdus. The nematodes are obligate parasites of insects that have an infective juvenile (IJ) stage, which seeks out a potential insect host to complete its life cycle. Harbored within the intestine of the IJ, Photorhabdus cells are released by regurgitation following entry of the nematode into the hemocoel of the insect (Ciche

& Ensign, 2003). The bacteria proliferate, killing the insect through the production of various toxins and exoenzymes and providing nutrients that optimize nematode growth and reproduction

(Forst & Clarke, 2002; Herbert & Goodrich-Blair, 2007)). Near the end of bacterial growth,

Photorhabdus produce antimicrobial compounds active against other bacteria, fungi, yeasts and nonhost nematodes (Akhurst, 1982; Boemare et al., 1997a; Webster et al., 2002) protecting the insect cadaver from colonization by other organisms. Once the food resources are exhausted, the

Heterorhabditis nematodes develop a new generation of IJs, which are colonized by the symbiotic Photorhabdus, and emerge from the insect cadaver in search of a new host. For the H. bacteriophora-P. luminescens mutualistic relationship, Ciche et al. (2008), have described the maternal transmission of the symbiont. The rectal glands of the maternal nematode are colonized by an axenic culture of P. luminescens, which is released into the lumen of the intestine by lysis of the gland cells. The IJ develops within the body cavity of the maternal nematode and is exposed to the symbiont. One cell adheres to the pharyngeal intestinal valve of the IJ and subsequently colonizes the intestine (Ciche et al., 2008).

Photorhabdus spp. are Gram-negative motile rods. They are facultative anaerobes, chemoorganotrophs with both respiratory and fermentative types of metabolism, negative for

oxidase, positive for catalase, and do not reduce nitrate to nitrite (Brenner & Farmer, 2005).

Most strains of Photorhabdus emit light and are the only known terrestrial bacteria to be bioluminescent (Gerrard, 2003), a trait that is gradually being lost (Peat et al., 2010). At present,

Photorhabdus is a member of the family Enterobacteriaceae, the only family of the order

Enterobacteriales, which consists of a variety of other symbiotic bacteria (Brenner & Farmer,

2005). However, a new family, Morganellaceae, has been proposed, which would include

Photorhabdus, its sister genus Xenorhabdus, along with the type genus Morganella and 5 other genera (Adeolu et al., 2016).

Currently, four recognized Photorhabdus species have been identified: P. luminescens

(Thomas & Poinar 1979; Boemare et al., 1993) (9 subspecies), P. temperata (Fischer Le-Saux et al., 1999) (6 subspecies), P. asymbiotica (Fischer Le-Saux et al., 1999) (2 subspecies) and P. heterorhabditis (Ferreira et al., 2014). The subspecies comprising P. luminescens are P. luminescens subsp. luminescens, P. luminescens subsp. akhurstii, P. luminescens subsp. laumondii (Fischer Le-Saux et al., 1999), P. luminescens subsp. kayaii, P. luminescens subsp. thracensis (Hazir et al., 2004), P. luminescens subsp. caribbeanensis, P. luminescens subsp. hainanensis (Tailliez et al., 2010), P. luminescens subsp. kleinii (An & Grewal, 2011) and P. luminescens subsp. noenieputensis (Ferreira et al., 2013b). The subspecies P. luminescens subsp. sonorensis has been proposed (Orozco et al., 2013) but has not yet been accepted. The subspecies included within P. temperata include P. temperata subsp. temperata (Fischer Le-

Saux et al., 1999), P. temperata subsp. cinerea (Tóth & Lakatos 2008), P. temperata subsp. stackebrandtii (An & Grewal, 2010), P. temperata subsp. khanii, P. temperata subsp. tasmaniensis and P. temperata subsp. thracensis (Tailliez et al., 2010). Due to its position in the phylogenic reconstruction of Photorhabdus using recA, gyrB, dnaN, gltX and rplB partial

sequences, P. luminescens subsp. thracensis was renamed P. temperata subsp. thracensis

(Tailliez et al., 2010). P. temperata subsp. stackebrandtii is a heterotypic synonym of P. temperata subsp. khanii (Ferreira et al., 2013b). P. asymbiotica (Fischer Le-Saux et al., 1999) is comprised of two subspecies: P. asymbiotica subsp. asymbiotica and P. asymbiotica subsp. australis, respectively isolated in the USA and Australia (Akhurst et al., 2004).

The Photorhabdus symbiont must be able to support nematode recovery, reproduction and development, and it must be retained within the heterorhabditid intestine (Han & Ehlers,

1998; Adams et al., 2006). Taxonomic data as well as gnotobiological studies reveal specificity between the nematode host and its bacterial symbiont (Boemare et al., 1997a; Han & Ehlers,

1998; Strauch & Ehlers, 1998). However, more than one species of Photorhabdus can be mutually associated with a species of Heterorhabditis. Photorhabdus luminescens is associated with most stains of H. bacteriophora as well as H. floridensis, H. georgiana, H. indica, H. mexicana, H. safricana, H. sonorensis, other Heterorhabditis spp. from China, Korea, St. Martin and Turkey as well as an undescribed nematode from France (Fischer Le-Saux et al., 1999; Hazir et al., 2004; Tailliez et al., 2010; An & Grewal, 2013; Ferreira et al, 2013b; Shapiro-Ilan et al.,

2014; Geldenhuys et al., 2016). Photorhabdus temperata is found associated with the nematodes

H. bacteriophora, H. downesi, H. georgiana, H. megidis, H. zealandica, Heterorhabditis spp. from Cuba, South Africa and Turkey and a nematode isolated in France that has not yet been identified (Fischer Le-Saux, 1999; Boemare, 2002; Akhurst et al., 2004; Tóth & Lakatos, 2008;

An & Grewal, 2010; Tailliez et al., 2010; Maneesakorn et al., 2011). Photorhbdus heterorhabditis is associated with H. zealandica in South Africa as well as Heterorhabditis sp.

Q614 from Australia (Ferreira et al., 2014). Until the discovery of a nematode host by Gerrard et al. (2006), P. asymbiotica had only been observed as clinical strains in human infections (Farmer

et al., 1988; Peel et al., 1999). The nematode host for P. asymbiotica Kingscliff strain, isolated in Australia, was similar to H indica and later described as a new species, H. gerrardi (Plichta et al., 2009). No host has been identified for P. asymbiotica isolated in the USA; however, in

Japan P. asymbiotica strains have been isolated from H. indica (Kuwata et al., 2008).

The 16S (small subunit) rRNA gene is often used for phylogenetic studies of prokaryotes.

However, this gene has some limitations. Like other bacterial genes, ribosomal genes can undergo lateral gene transfer across diverse taxonomic groups or be recombined, which could lead to misleading evolutionary data (Yap et al., 1999). Tailliez et al. (2010) demonstrated lateral transfer of 16S rRNA gene segments in Photorhabdus. Also, multiple copies of this gene exist within the bacterial chromosome. Consequently, resolving bacterial phylogeny based solely on 16S rRNA gene sequences may not be sufficient (Lerat et al., 2003). When analyzing

P. asymbiotica subsp. australis, Akhurst et al. (2004) found the DNA gyrase subunit B (gyrB) gene to be a more reliable marker than the16S rRNA gene for investigating Photorhabdus relationships at the species level. It has been used solely (Tóth & Lakatos, 2008; Shapiro-Ilan et al., 2009; An & Grewal, 2010; 2011; Shapiro-Ilan et al., 2014) and in conjunction with other housekeeping genes (Peat et al., 2010; Tailliez et al., 2010; Orozco et al., 2013; Ferreira et al.,

2013b; 2014; Blackburn et al., 2016) for reconstruction of Photorhabdus phylogeny.

The gyrB gene, which encodes subunit B of DNA gyrase is commonly used for bacterial taxonomy and phylogeny, and has many advantages. It is a single-copy gene, is of appropriate size and variability for phylogenetic, diversity, and taxonomic studies and putatively has low rates of horizontal gene transfer, and (Caro-Qintero & Ochman, 2015; Deng et al., 2014; Car-

Quintero et al., 2011). However, Photorhabdus trees constructed using the gyrB gene have shown some discordance with other housekeeping gene trees (Ferreira et al., 2014; 2013b).

Another gene that has been used successfully for determination of bacterial phylogeny within the family Enterobacteriaceae is the -subunit of RNA polymerase (rpoB) (Mollet et al.,

1997; Hoffman & Roggenkamp, 2003; Stephan et al., 2007; Behrendt et al., 2015). Like gyrB it is a single copy gene universally present in bacteria. The -subunit of RNA polymerase contains several variable regions, which are flanked by regions of conserved sequences (Lisitsyn et al.,

1988; Palenik, 1992). Mollet et al. (1997) found the hypervariable region 4 (Lisitsyn et al.,

1991) to be useful for phylogenetic studies among members of Enterobacteriaceae, and it has since been used for the identification and estimation of phylogeny among various other groups of bacteria (Adékambi et al., 2008).

In the context of studying native entomopathogenic nematodes indigenous to Florida, a survey was conducted throughout areas adjacent to operating citrus groves (Nguyen et al., 2006).

Several survey samples contained Heterorhabditis spp., and strains of bacteria were isolated from their respective nematode hosts. The objectives of this study were (a) to estimate genetic diversity within Photorhabdus populations native to Florida, (b) to reconstruct their phylogeny with known Photorhabdus spp., (c) to determine the utility of the rpoB gene as a marker for phylogenetic reconstruction of Photorhabdus, and (d) to investigate the involvement of recombination in these housekeeping genes.

Materials and Methods

Collection of Nematode Specimens and Isolation of Bacterial Strains

A survey was conducted throughout areas adjacent to operating citrus groves in search of entomopathogenic nematodes indigenous to Florida (Nguyen et al., 2006). Symbiotic bacteria were isolated from Heterorhabditis spp. obtained from the survey (Figure 2-1).

Entomopathogenic nematodes were extracted from soil samples using an insect baiting technique

(Bedding & Akhurst, 1975) and maintained in the laboratory on late instar larvae of the greater wax moth, Galleria mellonella L. For each soil sample, approximately 150 g of moist soil were placed into a 200 ml cup, and three late instar G. mellonella larvae were added to the soil surface. The cup was covered with a lid and incubated for 10 days at 24-25ºC. To collect emerging nematodes, dead larvae were removed from the soil, rinsed with sterile distilled water and placed on a modified White trap (Kaya & Stock, 1997). The trap consisted of a piece of moist 9.0 cm-diameter filter paper in an inverted Petri dish (60 × 15 mm) lid, on which the cadavers were placed, floating on a thin layer of water inside a larger Petri dish (150 ×20 mm).

Nematodes that migrated into the water were collected. All nematodes were identified to the species level by Dr. Khuong B. Nguyen, Department of Entomology and Nematology,

University of Florida.

Bacterial symbionts were isolated from entomopathogenic nematodes obtained from the survey as well as those maintained in the Nematode Evolution Laboratory at the University of

Florida, Gainesville representing a total of six identified species within the genus

Heterorhabditis. To isolate the symbiotic bacteria, infective juveniles were surface sterilized by incubating 30 min in a solution of 0.125% (w/v) methylbenzathonium chloride with gentle rocking, followed by a 15 min incubation in 3% (v/v) hydrogen peroxide. The IJ were triple rinsed with sterile tap water and macerated to liberate their symbiotic bacteria. Photorhabdus strains were identified based on morphological growth characteristics exhibited when cultured on

Tergitol-7 Agar (BBL, Becton Dickinson and Company, Franklin Lakes, NJ) supplemented with

0.004% triphenyltetrazolium chloride. Additional bacteria were provided by Dr. Ray Akhurst,

CSIRO Entomology, Canberra ACT 2601, Australia and Dr. David Bowen, Monsanto,

Chesterfield, MO. Photorhabdus asymbiotica subsp. asymbiotica strain 3265-86 ATCC 43950

was obtained from the American Type Culture Collection (Rockville, MD). Details of the origin and host affiliation of strains used in this study are presented in Table 2-1.

Preparation of Genomic DNA

Cultures were grown overnight at 27°C in 5 ml of Luria-Bertani broth with shaking at

150 rpm. The cells were collected by centrifugation of 1 ml of the broth culture at 8,000 rpm for

3 min. The resultant pellet was washed with 500 µl of sterile tap water, and the cells were again collected by centrifugation. Total genomic DNA was prepared by a modified method of Ausubel

(1992). Bacterial cells were incubated in 600 µl cell lysis buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 0.5% (w/v) sodium dodecyl sulfate; 30 µg proteinase K) for 1 h at 37°C. The DNA was purified from the cell lysates by incubation with 150 µl of 5 M sodium chloride and 80 µl

CTAB/NaCl solution (10% (w/v) cetyl trimethyl ammonium bromide in 0.7 M sodium chloride) for 10 min at 65°C followed by extractions with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1). The nucleic acids were precipitated with 0.6 vol isopropanol, washed twice with 70% (v/v) ethanol and dissolved in TE (10 mM Tris-HCl, pH

8.0; 1 mM EDTA). The DNA was quantified spectrophotometrically.

Repetitive Sequence-based Genotyping

Genomic fingerprints were generated for Photorhabdus strains using primers corresponding to the conserved BOX1A, REP and ERIC bacterial repetitive elements (Table 2-

2). Rep-PCR was performed as described by Louws and Cuppels (2001). Each reaction mixture contained 1 µl purified DNA (500 ng µl-1 for ERIC and REP-PCR or 50 ng µl-1 for BOX-PCR),

1 × Gitschier buffer (16.6 mM ammonium sulfate; 67 mM Tris HCl pH 8.8; 6.7 mM magnesium chloride; 6.7 µM EDTA pH8.8 and 30 mM ß-mercapto-ethanol), 10% (v/v) dimethyl sulfoxide,

4.0 mg bovine serum albumin, 1.25 mM each dNTP, 0.3 µg of each primer (only one primer used for BOX-PCR), 2 U AmpliTaq DNA polymerase (Life Technologies, Grand Island, NY),

and water to a final volume of 25 µl. The PCR amplifications were carried out in a PTC-100

Thermal Cycler (MJ Research, now Bio-Rad Laboratories, Hercules, CA) using the following thermal profile: initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at

94°C for 3 s and 92°C for 30 s, annealing at 40°C (REP-PCR) or 50°C (BOX- and ERIC-PCR) for 1 min and extension at 65°C for 8 min with a final extension step at 65°C for 8 min. The

PCR products were subsequently held at 4°C until they were resolved by gel electrophoresis.

PCR products were resolved by gel electrophoresis on 1.5% (w/v) agarose gels in 1× Tris- acetate-EDTA buffer. The gels were stained with ethidium bromide and photographed. A computer assisted pattern analysis was performed with the digital images using GelCompar software version 4.0 (Applied Maths, Sint-Martens-Latem, Belgium). The images were normalized using a 1 Kb DNA ladder (Invitrogen, Carlsbad, CA) that was loaded in first and every ninth well evenly dispersed throughout each agarose gel. Excessive background was subtracted by applying the rolling-disk option, and DNA bands detected by the software were confirmed by visual examination. A cluster analysis of the pairwise similarity values was performed using the unweighted pair group method using arithmetic averages clustering technique, and a dendrogram was constructed (Rademaker et al., 1999).

Amplification, cloning and sequencing of partial gyrB and rpoB genes

With the exception of strains isolated for this study, bacterial strains with a Pearson product moment correlation value of 60 or less based on profiles derived from rep-PCR were selected for phylogenetic analyses using the housekeeping genes gyrB and rpoB. The primers gyr-320, 5'-TAARTTYGAYGAYAACTCYTTAYAAAGT-3' and rgyr-1260, 5'-

CMCCYTCCACCARGTAMAGTTC-3' (R= G or A, Y = T or C and M = A or C) of Dauga

(2002) were used to amplify gyrB. Each reaction mixture had a total volume of 25 l containing

1 × PCR buffer, 2.5 mM MgCl2, 0.2 µM each deoxynucleoside triphosphate, 1 µM each forward and reverse primer, 1 U Taq polymerase (Promega Corporation, Madison, WI) and 1 l DNA template. The thermal cycling program consisted of an initial denaturation step for 3 min at

94C, 30 cycles of denaturation at 95C for 30 s, annealing at 53C for 1 min and extension at

72C for 2 min and a final extension step at 72C for 10 min.

Because of the high similarity among the gyrB sequences, fewer strains were selected for analyses using the rpoB gene. For the amplification of rpoB, primers were designed on the basis of sequence conservation in Photorhabdus luminescens subsp. laumondii TT01 (accession number BX571860), Escherichia coli O157:H7 EDL933 (AE005174), Salmonella enterica serovar Typhi CT18 (AL627279), Shigella dysenteriae Sd197 (CP000034) and Shigella sonnei

Ss046 (CP000038). The forward primer, rpF 5'-TGAGCCARTCTGGYCAYAARTCTAT-3', annealed at positions 974-998 (based on the numbering of the rpoB gene of P. luminescens subsp. laumondii TT01) and the reverse primer rpR 5'-TCACGRGAYACRCAMGCCAGTTC-3' annealed at positions 2503-2525 allowing for the amplification of the sequence encoding the variable polypeptide regions 3 through 5 and part of region 6 (Lisitsyn et al., 1988). Each reaction mixture had a total volume of 25 l containing 1 × PCR buffer, 2.5 mM MgCl2, 1 M betaine, 12.5 g bovine serum albumin, 0.2 µM each deoxynucleoside triphosphate, 0.5 µM each forward and reverse primer, 1 U Taq polymerase (Promega Corporation) and 0.5 l DNA template. The thermal cycling program consisted of an initial denaturation step for 3 min at

95C, 30 cycles of denaturation at 94C for 30 s, annealing at 54C for 1 min and extension at

72C for 2 min and a final extension step at 72C for 10 min.

All PCR was performed using a PTC-200 DNA Engine peltier thermocycler (MJ

Research, now Bio-Rad Laboratories). For each reaction, a 5l vol of PCR product was

electrophoresed on 0.7% (w/v) agarose containing ethidium bromide (10 mg ml-1) and visualized under UV light to ensure the amplicon was the correct size. Amplicons were purified using the

QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA) and directly sequenced using the forward and reverse PCR primers. For sequencing of the rpoB gene, the internal forward primer

RpoIF 5'-GTATGTCCWATCGAAACSCCTG-3' (W = A or T and S = G or C), annealing to positions 1672-1693, and the internal reverse primer RpoIR 5'-

CTTCAGGSGTTTCRATWGGAC-3' (R = A or G), annealing to positions 1676-1696, were designed as described above. These primers along with the forward primer of Mollet et al.,

(1997) CM7 5'-AACCAGTTCCGCGTTGGCCTGG-3', annealing to positions 1384-1405, were used to sequence the internal portion of the gene product.

When direct sequencing failed, which only occurred for the gyrB product, the amplicons were ligated into a T-overhang vector using the pGEM®-T Vector System (Promega), which was then transformed into MAX Efficiency® DH5α™ Competent Cells (Invitrogen). The presence of cloned inserts was verified by colony-PCR. Each colony was picked with a sterile toothpick and transferred to the surface of an LB agar plate containing ampicillin. A second toothpick placed cells from the colony into thin-walled tube containing 45 µl of sterile distilled water. The cells were lysed by boiling for 7 min and collected by centrifugation at 10,000 x g for

10 min. The supernatant (10 µl) was withdrawn and used for the template. Each reaction mixture (25 l total vol) contained 1 × PCR buffer, 1.5 mM MgCl2, 0.2 µM each deoxynucleoside triphosphate, 0.2 µM each primer (either T7 and Sp6 promoter primers or T7 promoter primer and reverse PCR primer) and 1 U Taq polymerase (Promega Corporation). The thermal profile consisted of an initial denaturation of 95C for 3 min, followed by 35 cycles of denaturation at 94C for 1 min, annealing at 50C for 1 min and extension at 72C for 2 min and

a final extension step at 72C for 10 min. Insert-containing colonies were grown overnight in

LB broth containing ampicillin and purified using the QIAprep Spin Miniprep Kit (Qiagen, Inc.).

Purified gene products were sequenced using the ABI BigDye® Terminator v1.1 cycle sequencing kit (Life Technologies) and a modified protocol. Each sequencing reaction contained

2.0 l BigDye® ready reaction mix, 2.0 l ICBR mix (proprietary, University of Florida), 3.2 pM sequencing primer, 15 ng (PCR product) or 350 ng (plasmid) template and purified water to a final reaction volume of 10 l. The following thermal profile was used for amplification: denaturation at 96C for 30 s, primer annealing at 50C for 15 s and product extension at 40C for 4 min for a total of 25 cycles and then held at 4 C. The sequencing products were purified by ethanol precipitation. Each sequencing reaction product was placed into a 1.5 ml microcentrifuge tube containing 30.0 l of cold 95% ethanol and 1.0 l 3 M sodium acetate, pH

5.2, incubated on ice for 10 min and centrifuged at 12,000 x g for 15 min. The ethanol mixture was removed, and the resulting pellet was rinsed with 250 l of 70% ethanol and dried using a rotary evaporator. Purified sequencing reaction products were recorded at the University of

Florida ICBR DNA Sequencing Facility, Gainesville, FL with an ABI 3130 automated DNA sequencer (Life Technologies). All gene sequences were submitted to GenBank (Accession numbers HQ667713-HQ667760).

Phylogenetic Analyses

Gene sequences were assembled and edited using Sequencher software (Gene Codes

Corporation, Ann Arbor, Michigan). Sequence data for gyrB and rpoB were obtained from

GenBank (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/genbank/) for all other taxa (Table 2-3). Nucleotide sequences were aligned using ClustalW 2 (Thompson et al., 1997) on the EMBL-EBI web server

(http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/) (Larkin et al., 2007; Goujon et al., 2010) and then optimized manually using MacClade 4.08 (Maddison & Maddison, 2005) based on their translated amino acids. For taxa with concatenated gyrB-rpoB sequences that were identical, all taxa but one were removed from the alignment. Aligned sequences were converted to FASTA and NEXUS file formats using ALTER (Glez-Peña et al., 2010). Both genes were analyzed individually and together as a concatenated dataset. There were a total of 6 datasets based on the availability and size of gene fragments in GenBank: three for gyrB consisting of 48, 47 and 23 ingroup taxa, two for rpoB each consisting of 23 ingroup taxa and one combined dataset of 23 ingroup taxa. The gyrB 48 taxa dataset was for constructing a phylolgeny for Photorhabdus that represented strains of all known subspecies. The gyrB 47 taxa dataset was used for recombination analyses; one taxon was deleted to have fewer sequences of shorter length. The gyrB 23 taxa dataset consisted of taxa that corresponed with the rpoB 23 taxa datateset, and the other rpoB 23 taxa dataset had an additional P. heterorhabditis strain and one

P. asysmbiotica strain was removed due to redundancy.

Equally weighted parsimony analysis was conducted to find a minimum-length tree

(heuristic search, step-wise addition, addition sequence = as is, tree bisection reconstruction

(TBR) branch-swapping algorithm) using PAUP v4.0b10 (Swofford, 2002). Gaps were treated as missing data. Branch support was estimated by bootstrapping using the same tree search parameters (1000 replicates). To test for phylogenetic congruence between gyrB and rpoB partial gene sequences, the statistical significance of the incongruence length difference (ILD;

Farris, et al., 1994, 1995) or partition homogeneity was assessed in PAUP v4.0b10 (Swofford,

1998) by executing 1000 replications. To confirm these findings, the Templeton test (two-tailed

Wilcoxon signed-rank test) (Templeton, 1983) also implemented in PAUP v4.0b10 (Swofford,

2002) was used to test for congruence between the most parsimonious trees for each data partition.

Prior to Bayesian and maximum likelihood analyses, models of sequence evolution were evaluated using JModelTest 2.1.7 (Guindon & Gascuel, 2003; Darriba et al., 2012) with Akaike weights (Posada & Buckley, 2004) for each gene partition. For Bayesian analysis implemented in MrBayes, three substitution schemes were used to test for the best of 24 models. For maximum likelihood analyses, 11 substitution schemes were used to test for the best of 88 models or 203 substitution schemes were used. The best fit models of nucleotide substitution chosen by JModelTest are presented in Table 2-4 or otherwise presented in the legend of their respective figure.

Bayesian analyses were implemented in MrBayes 3.2.6 (http://mrbayes.sourceforge.net)

(Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003). Two Markov chain Monte

Carlo (MCMC) analyses were run for each datasets with four chains (one cold and three hot).

Each analysis ran for 10 million generations (printfreq=1000). After the analyses were completed, the application Tracer 1.4 (Rambaut & Drummond, 2014) was used to verify that stationarity had been reached. Trees generated prior to reaching stationarity were discarded at burn-in. Maximum likelihood analyses were conducted using GARLI 2.01, Genetic Algorithm for Rapid Likelihood Inference (http://code.google.com/p/garli/downloads/list) (Zwickl, 2006), applying the models of evolution selected by JModelTest2. Five search replicates were performed from random starting trees, and the best scoring tree was selected. The bootstrap analyses were run using two search replicates from random starting trees and 1000 bootstrap replicates. The majority-rule consensus trees were calculated using the program SumTrees v4.0

of the DendroPy v4.0.3 package (Sukumaran & Holder 2010). Both MrBayes and GARLI analyses were conducted using the CIPRES Science Gateway (Miller et al., 2010).

Recombination Analyses

Occurrence of recombination in gyrB and rpoB was first investigated by the construction of split decomposition graphs and the calculation the pairwise homoplasy index (PHI) (Bruen et al. 2006) using SplitsTree4 v4.14.4 (http://splitstrees.org) (Huson & Bryant, 2006). To search for recombinant breakpoints in the alignments, the phylogenetic method GARD (Genetic

Algorithm for Recombination Detection) (Kosakovsky Pond et al., 2006) was implemented using the Adaptive Evolution Server (www.datamonkey.org) (Delport et al., 2010). The

Tamura-Nei (TrN) model was selected using the local model selection program along with the β-

Γ site-to-site rate variation and a rate class of 4. To further assess recombinant breakpoints in the alignments, seven nonparametric detection programs were implemented using the RDP4,

Recombination Detection Program version 4.35 (Martin et al., 2010). Of these, Bootscan

(Martin et al., 2005) and RDP (Martin & Rybicki, 2000) are phylogenetic; SiScan (Gibbs et al.,

2000) uses a distance based method; Chimaera (Posada & Crandall, 2001), GENECONV

(Padidam et al., 1999) and MaxChi (Maynard Smith, 1992) use substitution methods; and 3SEQ

(Boni et al., 2007) uses hypergeometric random walks. Each program was run on both single gene and concatenated alignments. For highly similar sequences all sequences but 1 with an uncorrected distance smaller than 0.0011 (gyrB) and 0.0060 (rpoB) were removed from each alignment. All sequences were considered circular. The highest acceptable P value cut-off was set to 0.01 for gyB and 0.05 for rpoB (to explore for more possible recombination events), a

Bonferroni correction was applied, and breakpoints were polished. For both Chimaera and

MaxChi 1000 permutations were generated.

Results

Isolation of nematodes

In the survey of nematodes that was conducted in areas adjacent to active citrus groves,

308 samples were taken of which 26 samples (8.4%) contained entomopathogenic nematodes.

Twenty-two of the samples (7.1%) contained Heterorhabditis spp. (Nguyen et al., 2006).

Morphological and molecular studies showed that most Heterorhabditis isolates were H. indica

Poinar, Karunaka & David, 1992, one isolate from the survey was H. zealandica Poinar, 1990 and another was H. floridensis Nguyen, Gözël, Koppenhöfer and Adams, 2006. Fourteen of the

22 isolates of Heterorhabditis spp. were selected for this study, including 12 isolates of H. indica along with the H. zealandica and H. floridensis isolates.

Diversity as Assessed by rep-PCR

Genomic fingerprints were generated from total chromosomal DNA extracted from 55 strains of Photorhabdus (6 were from the same strain of nematode but from different sources) and four non-pigmented forms thereof (Table 2-1). For the combined BOX, REP and ERIC amplified sequences; complex patterns were formed for each strain consisting of 32 or more

DNA fragments that ranged in size from approximately 0.26 to more than 8.5 kb. Pearson’s product-moment correlation (r) values were calculated for the DNA fingerprints revealing distinct clusters for P. asymbiotica subsp. asymbiotica, P. heterorhabditis and P. luminescens at the level of subspecies (Fig. 2-2).

The bacterial strains isolated from H. indica from the survey all clustered with other strains of P. luminescens subsp. akhurstii as expected. Within this cluster, the Florida strains separated into 2 distinct groups, and strain FL406, being least similar to the other strains, did not converge with either cluster. Most of the strains converged together along with a strain isolated from H. indica from Georgia, whereas strains FL265, FL354 and FL479 formed a cluster with

bacterial strains isolated from H. indica from Saudi Arabia, and FL406 was independent of both groups. Strain FL480 clustered with the other strains isolated from H. zealandica generating the most unique profile for this group. Finally, FL332 was part of the P. luminescens subsp. luminescens cluster. This cluster was comprised of bacteria isolated from H. bacteriophora, H. mexicana and H. floridensis.

Photorhabdus temperata subsp. temperata strains merged into one group, and the representative strain of P. temperata subsp. tasmaniensis, MegDB, converged with this group.

Whereas unique genomic profiles were generated for representative strains of P. temperata subsp. khanii (synonym P. temperata subsp. stackebrandtii) NC19 and Mar1 and P. temperata subsp. cinerea Pjun. Though the P. t. khanii strains were within the same larger cluster, the

Pearson product moment correlation value was just over 20. Strain Mar1 was located within the

P. a. asymbiotica cluster and strain NC19 was the most divergent of the group. P. t. cinerea converged with the P. heterorhabditis cluster. With all three strains and Photorhabdus t. tasmaniensis, the linearly combined BOX, REP and ERIC fingerprints did not produce significant similarity (<40) to those generated by the other Photorhabdus strains presented in this study.

The non-pigmented forms of the four isolates (P. asymbiotica subsp. asymbiotica 3265-

86, P. luminescens subsp. laumondii HP88 and HP88A and P. temperata subsp. khanii NC19) clustered tightly together with their clonal strains, each with a similarity value greater than 90.

For P. a. asymbiotica 3265-86 and P. t. khanii NC19, the genotypic patterns appeared identical.

For P. l. laumondii HP88 and HP88A, the patterns were the same for those amplified using REP and ERIC primers; however, both of the non-pigmented forms produced and extra band of about

5.4 kb when using the BOX primer.

gyrB and rpoB based Phylogenetic Analyses

Phylogenetic analyses were performed using partial gene sequences consisting of 918 and

1447 nucleotides for gyrB and rpoB, respectively. Most of the Florida strains from H. indica were indistinguishable using only the gyrB sequence. The sequences for strains FL237, FL265,

FL301, FL304, FL319, FL370 and FL482 were identical to each other and to P. luminescens subsp. akhurstii strain K81 (AY278510). Strain FL480 was identical to strains ENY and ZM isolated from H. zealandica from FL and SC, respectively as well as to P. heterorhabditis strains

SF41 (KF418144), SF783 (KF418150) and VMG (NZLJCS01000024). For the rpoB partial sequences FL304, FL319 and FL482 were identical. Once identical gene sequences were removed from the alignments, the similarity among nucleotide sequences of the Photorhabdus spp. strains presented here ranged from 86.2 to 99.9% for gyrB and 91.6 to 99.9% for rpoB.

Variability among nucleotides within the aligned sequences is indicated in Figure 2-3. The final alignments for the gyrB datasets contained 259 (48 taxa) and 242 (23 taxa) parsimony informative sites and 263 (25 taxa) and 255 (23 taxa) parsimony informative sites for the rpoB datasets.

The partition homogeneity showed significant incongruence between the phylogenies for gyrB and rpoB (P = 0.1) and the results of the Templeton test rejected congruency between the two topologies (P ≤ 0.0001). For this study, phylogenies were inferred using gyrB and rpoB sequences individually and concatenated. A maximum likelihood phylogenetic reconstruction using gyrB for the 48 ingroup taxa dataset is presented in Figure 2-4. Where concordant with the liklelihood tree, the bootstrap support values for maximum parsimony and the posterior probability values as percentages obtained from MrBayes that are above 70 are presented.

Similar trees were produced by each of the three methods with the exception of the clade containing P. heterorhabditis and P. asymbiotica. For maximum likelihood, the tree bifurcates

into 2 clades: one clade with only P. luminescens and the other clade with P. asymbiotica, P. heterorhabditis and P. temperata. For the Bayesian tree, one clade contains P. luminescens, P. asymbiotica and P. heterorhabditis, while the other clade contains only P. temperata. All of the strains isolated for this study with the exception of FL480 appear in the P. luminescens clade.

The strains isolated from H. indica nematodes are monophyletic with P. luminescens subsp. akhurstii as expected based on their nematode host and their close nucleotide similarity. The closest sister taxon is P. luminescens subsp. hainanensis. The strain FL480 is monophyletic with

P. heterorhabditis, which is sister to P. asymbiotica.

In Figure 2-5, a maximum likelihood tree is derived from rpoB sequences. In this tree the clade containing FL480 is again part of a taxon group that is sister to P. asymbiotica that is more closely related to P. luminescens than to P. temperata. Also, the P. heterorhabditis clade forms a clade with P. t. cinerea Pjun. In the tree constructed with gyrB, P. t. cinerea Pjun is in the P. temperata clade with P. t. cinerea. Photorhabdus asymbiotica subsp. australis, does not converge with P. asymbiotica subsp. asymbiotica, but is sister to P. luminescens akhurstii.

Figure 2-6 shows a comparison of maximum likelihood trees based on the 23-taxon datasets of gyrB and rpoB. The trees have similar topology with the exception of the position of

P. heterorhabditis, P. asymbiotica subsp. australis and P. t. cinerea Pjun. The trees demonstrate similar phylogenies: however; unlike the gyrB tree reconstruction, all groups were resolved at the level of species and subspecies for the rpoB tree. Figure 2-7 is the maximum likelihood tree of the concatenated sequences. The tree is similar to the phylogeny derived from rpoB sequences in that the P. asymbiotica clade is closer to P. luminescens than to P. temperata, and P. temperata diverges from the rest of Photorhabdus. Although, P. asymbiotica, P. heterorhabditis and P. t. cinerea Pjun do not form a single clade, but rather have separate lineages, and P. a. australis

converges with P. a. asymbiotica. All sequences are resolved at the level of subspecies. When

P. t. cinerea Pjun and P. a. australis were removed from the concatenated dataset, a partition homogeneity test indicated the phylogenies for gyrB and rpoB to not be significantly different (P

= 0.34).

Recombination

All methods used suggest the occurrence of recombination. Split decomposition graphs were constructed for individual as well as the concatenated gene sequences. Due to the low fit index (38.912) when all taxa were combined, the split decomposition graphs are presented as two separate graphs for gyrB. A split decomposition graph for P. luminescens and a graph for all other Photorhabdus from this study are depicted in Figure 2-8 A and B, respectively. The split decomposition graph for P. luminescens is more tree-like than that of the other Photorhabdus strains, but there are parallel edges for P. l. luminescens and two polytomies near the center of the graph (P. l. caribbeanensis and P. l. hainanensis). For the other Photorhabdus strains, the split decomposition graph indicates that P. heterorhabditis is close to P. asymbiotica forming a group and P. temperata is divergent from the two other species. The graph is reticulate in reference to the relationships of P. t. cinerea Pjun and P. t. thracensis to the other strains. The split decomposition graph for rpoB is illustrated in Figure 2-9. The graph consists of 23 taxa that are divided into 3 groups. The group containing P. heterorhabditis and P. a. aysmbiotica has a thin box representing some conflict in the phylogeny, as do the parallel edges leading to the group containing P. a. australis and P. luminescens. Figure 2-9 is a split decomposition graph of the concatenated gyrB-rpoB sequences. The graph indicates discordance among relationships within the group containing P. heterorhabditis and P. asymbiotica and conflicting phylogenic signals with their relationship to P. temperata.

The pairwise homoplasy index (PHI) presented in Table 2-5 found significant evidence for recombination when using all strains of Photorhabdus for gyrB and rpoB. The PHI (P = 0.0) for the concatenated sequences is in strong support of the reticulations presented in the Figure 2-

9. The three major clades that formed in the maximum likelihood trees were screened separately for recombination using the PHI test. Within the P. luminescens clade, there was no evidence of intragenic recombination in gyrB or rpoB, but there was evidence of intergenic recombination (P

= 0.00603). For the P. asymbiotica-P. heterorhabditis and P. temperata clades the PHI test rejected the hypothesis of no recombination for gyrB, implying intragenic recombination within their respective clades. Also, there was no evidence of recombination within rpoB or the concatenated sequences. These data suggest that recombination could be occurring between species.

Analyses of alignments of both the gyrB and rpoB genes using GARD and RDP4 suggest that both sequences show occurrences of recombination events in Photorhabdus. The putative breakpoints as determined using GARD are presented in Table 2-6. A single breakpoint was detected for gyrB, which occurs at either position 490 or 499 in the 918-nucleotide alignment. A single breakpoint was detected for rpoB at position 724 of the 1447 -nucleotide alignment, and from another analysis two breakpoints were detected at positions 328 and 730. All breakpoints were found repeatedly during multiple runs, and at times did not show up. The phylogenetic trees produced from the alignments to the right and to the left of the breakpoints had significantly different topologies (Table 2-6).

Once breakpoints had been detected using GARD, the alignments were examined using

RDP4 to confirm these breakpoints or to look for the possibility of other recombination events.

The breakpoints and the methods used to detect them within the RDP4 package are presented in

Table 2-7. For gyrB, the first recombination event detected with the highest probability occurred in the P. l. kleinii strain as detected by all 6 methods. The major parents were identified as most of the P. luminescens strains from the dataset (P. l. kayaii, P. l. akhurstii, P. l. laumondii, P. l. hainanensis and P l. caribbeanensis), and the minor parents were identified as 3 species of P. temperata (P. t. temperata, P. t. khanii and P. t. thracensis). Another event involved P. t. cinerea Pjun and P. t. cinerea 3107 as the potential recombinants with the area of suspected recombination occurring between 23 and 534 in one scenario or between 911 and 534 in the other. The potential parents were determined to be the same P. temperata species from the previous event and P. heterorhabditis. Finally, a smaller event, which only covered 115 bases, was detected in P. heterorhabditis Q614. The major and minor parents were identified as P. heterorhabditis and P. a. australis, respectively.

For rpoB two events were detected, one of which may not be due to recombination. The first event occurred between bases 148 and 824 of P. t. cinerea Pjun. The minor parent was identified as P. a. asymbiotica and the major parent as P. heterorhabditis. For this event one of the parents could actually be the recombinant strain. The second event occurred between bases

1414 and 640, and may be due to another evolutionary event besides recombination. The putative recombinant is P. l. laumondii (TT01 and HP88), and P. heterorhabditis was detected the minor parent. The major parent is unknown but possibly P. temperata Mar1. Because not many events were detected within rpoB and none concerning P. a australis, the maximum P- value was set to 0.05. An event was detected between positions 379 and 976 where P. a australis was the recombinant. The major and minor parents were determined to be P. l. akhurstii and P. a. asymbiotica respectively. This event was only significant with Maxchi (P =

0.04).

When the concatenated alignments were evaluated in RDP4 only one event occurred across both genes. This event was significant by all methods and occurred between 2332 and

534. The recombinant strain was P. t. cinerea Pjun, and the major and minor parents were P. heterorhabditis and the 3 species of P. temperata, respectively, as determined with gyrB. An event was detected in which P. a. australis was the recombinant and involved 708 bases of the gyrB gene. The minor parent was determined to be P. heterorhabditis and the major parent P. l. akhurstii. Photorhabdus heterorhabditis (VMG and FL80) was identified in a recombination event that spanned 715 bases of the rpoB gene. The minor parent was P. l. laumondii and the major parent P. t. cinerea Pjun. One of the parents could potentially be the recombinant.

Finally, a small region of only 46 bases was detected in the rpoB gene portion of P. l. akhurstii

(FL406 and FL265) but may be due to another evolutionary event.

Regions of the alignments on either side of potential breakpoints for gyB and rpoB were subjected to the PHI to evaluate if there was still significant evidence for recombination. The

PHI P –values are presented in Table 2-8. Few regions were determined to have no significant evidence of recombination.

Figures 2-11 – 2-14 illustrate the differences in tree topology obtained from analyses of alignments from different sides of the breakpoints. In Figure 2-11, trees were constructed with different portions of the gyrB alignment according to breakpoints determined in RDP4. Tree A

(PHI, P < 0.05), shows P. t. cinerea to be sister to P. heterorhabditis; while in tree B (PHI, P >

0.05), P. t. cinerea is sister to P. temperata. Also, tree B demonstrates better resolution among the other strains, especially where in tree A P. l. kleinii is part of the P. temperata clade.

In Figure 2-12 A not only does the position of P. t. cinerea change, but also the position of P. heterorhabditis Q614 and P. a asymbiotica. However, only the position of P. t. cinerea

with P. heterorhabditis using the second portion of the gyrB has good support. Figure 2-12 B demonstrates that if the putative recombination region is removed from P. l kleinii, it forms a monophyly with P. l. kayaii with strong support in the P. luminescens clade.

Alternative topologies based on different regions of the rpoB gene are illustrated in

Figures 2-13 and 2-14. In Figure 2-13 tree A, constructed with nucleotides 1-378 and 977-1447

(PHI, P < 0.05), P. a. australis is located outside of P. l. akhurstii. Tree B, nucleotides 379-976

(PHI > 0.05), places P. a. australis with P. a asymbiotica, which is the sister clade to P. heterorhabditis. For tree A in Figure 2-14, nucleotides 1-147 and 825-1447(PHI, P < 0.05), P. t. cinerea Pjun is within the P. heterorhabditis clade with strong support. For tree B (PHI, P >

0.05) P. t. cinerea Pjun forms a clade with P. a. asymbiotica, but the support is not strong.

Figure 2-15 is maximum likelihood tree constructed with the concatenate gyrB-rpoB genes with the recombination region removed. The tree is fully resolved and bifurcates into two clades, one with P. luminescens, P. asymbiotica and P. heterorhabditis and the other with P. temperata.

Discussion

Rep-PCR has proven to be an excellent and efficient method for the differentiation of various bacteria (Louws et al., 1994, 1998; Hahn et al., 2007; Tailliez et al., 2006) and the identification of clonal strains (Hoffman & Roggenkamp, 2003; Ramette &Tiedje, 2007).

Rademaker et al. (2000) were able to demonstrate congruence between rep-PCR and DNA:DNA hybridization, and it has been suggested that this could be an alternative for the more tedious and difficult DNA reassociation method for species delineation (Stackebrandt et al., 2002). This is the first study to demonstrate the utility of rep-PCR in Photorhabdus, and it showed that rep-

PCR with concatenated profiles derived from BOX, REP and ERIC primers was able to distinguish most strains used in this study at the level of subspecies.

All of the Photorhabdus strains retrieved from the Florida H. indica isolates demonstrated uniformity among the genetic fingerprints generated by rep-PCR (Figure 2-2). All strains from H. indica formed one group, which also included strains from Georgia (USA), India and Saudi Arabia. For the Florida strains, these profiles along with the gyrB partial sequences confirmed that they are all strains of P. luminescens subsp. akhurstii. The rpoB partial sequences for FL265, FL304, FL319, FL354, FL370, FL406 and FL482 further confirmed this.

The Florida strains were subdivided into three genomic profiles by rep-PCR (A-C).

Several strains were selected for sequencing of the partial gyrB gene: two from group A, six from group B and strain FL406 (profile C). The sequences generated from group B were identical to each other and to that of FL265, though the similarity between these two genomic profiles was less than 60% by rep-PCR. The gyrB sequences were highly similar among strains,

98.6-99.9%. The rpoB gene sequences were also highly conserved among the Florida strains

(99.6-100%) and other P. l. akhurstii strains (99.3-99.9%). In most cases the natural symbiont of

H. indica is P. l. akhurstii (Fischer-Le Saux et al., 1999; Nguyen, 2007), though H. indica has been found to associate with P. asymbiotica in Japan (Kuwata et al., 2008). All symbionts from

H. indica presented here are P. l. akhurstii.

Shapiro-Ilan et al. (2014) identified the FL332 strain along with another strain from H. floridensis K22 from Georgia (USA) as P. luminescens subsp. luminescens. Strain FL332 clustered with the other P. l. luminescens strains, which consisted of strains from H. floridensis,

H. mexicana and an undescribed Heterorhabditis sp. from South Carolina. Strain FL332 specifically converged with the ENY25 isolate of H. floridensis from Gainesville, FL. The data presented here support the findings of Shapiro-Ilan et al. (2014).

The type location of H. zealandica is New Zealand, but it is also native to Florida

(Nguyen, 2007; Stewart et al., 2015) as well as Lithuania, Russia, Australia and South Africa

(Hominick, 2002; Malan et al., 2006). Until recently the only bacterial symbiont associated with

H. zealandica had been identified as P. temperata subsp. tasmaniensis (Tailliez et al., 2010;

Ferreira et al., 2014). Ferreira et al. (2014) described a new species, P. heterorhabditis, associated with H. zealandica from South Africa and Heterorhabditis sp. strain Q614 from

Australia. The data presented here indicate that the bacterial symbiont of H. zealandica native to

Florida is P. heterorhabditis. The gyrB sequences from strains ENY and NCZ isolated in

Gainesville, FL and South Carolina, respectively, indicate that these strains are also P. heterorhabditis. All strains except strain FL480 produced nearly identical genomic fingerprints with a similarity value greater than 90%. Vinuesa et al. (2005) used a conservative threshold of r

> 80% to identify potential clone mates among Bradyrhizobium species. Accordingly, the data presented here suggest that all these strains are P. heterorhabditis. With the strains isolated thus far that were typed for this study (6 nematodes isolates), it appears the H. zealandica native to the US is associated with P. heterorhabditis and not P. t. tasmaniensis. Photorhabditis t. tasmaniensis is associated with H marelata Liu & Berry, 1996, (Tailliez et al., 2010), a nematode that has been isolated from the west coast of the US in Oregon (Liu & Berry, 1996) and California (Stock et al., 1999).

With rep-PCR, when the similarity of the genomic profiles is < 65%, saturation causes less reliability for the determination of relationships at a higher taxonomic level (Rademaker et al., 2000; Vinuesa et al., 2005; Ramette& Tiedje 2006). This was demonstrated here.

Photorhabditis luminescens subsp. laumondii did not group with the other members of P. luminescens but was more closely related to P. asymbiotica, while strains of P. temperata were

interspersed between other species lineages. Though the two representative strains from P. temperata subsp. khanii (Mar1 and NC19) formed a monophyletic group with both gyrB and rpoB sequence analyses, their genomic profiles were not similar and did not converge. However, when the global alignment setting was used instead of fine alignment, Mar1 and NC19 did form a single cluster but FL354 and P. a. asymbiotica 3105-77 were displaced (data not shown).

More sequences representing P. t. khanii, P. t. tasmaniensis, and P. temperata subsp. cinerea should be examined. This technique provided a fast and accurate method for determining which bacteria were most closely related so that redundant sequencing could be avoided during phylogenetic analyses. All the bacterial strains isolated for this study clustered with known species/subspecies suggesting that none of the species isolated from the survey are novel. This finding was further supported by the phylogenetic analyses.

It has been noted that 16S rDNA alone does not provide good characterization of bacterial relationships among Photorhabdus species (Akhurst et al., 2004; Tailliez et al., 2010), although Peat et al. (2010) did find that small subunit RNA, when used in combination with housekeeping genes, is useful for resolving closely related strains. Partial gene sequences for

DNA gyrase B have been largely used to described new Photorhabdus species and subspecies individually (Akhurst et al., 2004; An & Grewal, 2010; 2011; Tóth and Lakatos, 2008) and in combination with other genes (Tailliez et al., 2010; Orozco et al., 2013; Ferreira et al., 2013b;

2014). It has also been used in combination with other genes to investigate Photorhabdus evolutionary relationships (Peat et al., 2010; Blackburn et al., 2016). An objective of this study was to find an alternative gene sequence that would be sensitive enough, yet not have too many variable nucleotides, to distinguish species of Photorhabdus. The gene for rpoB has been used extensively for characterization of bacterial strains and the building of phylogenies for other

enteric species (Mollet et al., 1997, Adekambi et al., 2008; Behrendt et al., 2015). It was also selected by Lerat et al. (2003) as a gene within the γ-proteobacteria that has a low probability of lateral gene transfer.

This is the first study to demonstrate the utility of rpoB as a marker for Photorhabdus phylogeny. It was more sensitive than gyrB for distinguishing the Florida strains. This could be due to the larger gene region amplified, which allowed for more phylogenetically informative characters. An interesting point observed among the phylogenetic reconstructions presented here were the positions of P. heterorhabditis and P. asymbiotica. In the gyrB generated trees these species were more closely related to P. temperata than to P. luminescens. In most other published phylogenetic reconstructions using gyrB, the position of P. asymbiotica is monophyletic with P. luminescens with strong support values (Shapiro-Ilan et al., 2009; 2014;

Peat et al., 2010; Ferreira et al., 2013b; 2014; Blackburn et al., 2016). Most of these studies used gyrB sequences concatenated with other genes, so so it is possible that this phylogenetic signal was coming from the other gene sequences. However, the trees presented by Shapiro-Ilan et al. (2009, 2014) were based solely on the gyrB sequence. An alternative topology presented by Orozco et al. (2013) constructed from the analysis of the concatenated sequences of gyrB with dnaN, glxX and recA shows P. asymbiotica to be more distantly related to both P. luminescens and P. temperata. The trees produced by the analyses of rpoB were in better support of previously published phylogenies (Peat et al., 2010; Tailliez et al., 2010; Ferriera et al., 2013b;

2014). Photorhabdus heterorhabditis and P. asymbiotica formed a monophyletic group that was sister to P. luminescens, but P. asymbiotica subsp. australis converged with the P. l. akhurstii clade and not P. asymbiotica subsp. asymbiotica.

The ILD indicated these genes were discordant, and the discordance between

Photorhabdus gyrB and rpoB is illustrated in Figure 2-6. However, it has been argued that data yielding discordant phylogenetic reconstructions may be analyzed together (Cunningham, 1997), and that the ILD may not be an accurate test for data combinability (Yoder et al., 2001). In an attempt to dilute any artificial phylogenetic signal, the two genes were combined for phylogenetic analyses. The relationship of P. a. australis with P. a. asymbiotica was recovered, but some support for other bacterial evolutionary relationships was lost, which included the positions for P. heterorhabditis and P. t. cinera .

Although gyrB has not demonstrated lateral gene transfer in Photorhabdus (Tailliez et al.,

2010; Ferreira et al., 2013b) and rpoB is considered a gene to have a low probability of lateral gene transfer (Lerat et al., 2003), the gene alignments were investigated for recombination to determine if this could be implicated for the conflicting topologies. Homologous recombination is a driving force of bacterial evolution, but this exchange of genetic information can lead to false phylogenies (Posada & Crandall, 2002).

The data presented here suggest the occurrence of both intergenic and intragenic recombination. In the split decomposition graphs (Figures 2-8 – 2-10), the observation that multiple pathways forming an interconnected network rather than a single bifurcating tree linking the Photorhabdus strains to each other is often indicative of recombination (Holmes et al., 1999). This was clearly illustrated in Figures 2-8A and 2-10 and supported by the PHI values. The relationships presented here are in agreement with the maximum likelihood and

Bayesian phylogenies.

The three phylogenetic groups, P. luminescens, P. temperata and P. asymbiotica + P. heterorhabditis, were investigated individually and together. Within P. luminescens, gyrB and

rpoB do not appear to undergo intragenic recombination; rather the data suggest that intragenic recombination occurs with other species. Using the gltx gene, Tailliez et al. (2010) showed that a P. luminescens subsp. kayaii sequence was a mosaic consisting of sequence regions from other

P. l. kayaii strains and a region from P. temperata subsp. thracensis. Their research also showed evidence of recombination within the 16S rRNA gene of two P. l. kayaii strains with other strains of P. l. kayaii and P. t. temperata (Tailliez et al., 2010). If one were to examine these genes individually only in species of P. luminescens, then recombination may not be evident; however, when the genes were concatenated there was evidence of intergenic recombination.

For P. temperata, intrageneic recombination of gyrB appeared to occur within and across species. This was also true for the monophyletic group of P. asymbiotica and P. heterorhabditis.

The portion of the gene used for phylogenetic analyses had immense impact on the topologies of the trees. Trees were constructed with sequences on either side of putative breakpoints yielding very different topologies. For the gyrB gene, if the tree was constructed with nucleotides from the later part of the sequence, P. t. cinera converged with P. heterorhabditis to form a monophyletic clade whose closest sister taxa were P. asymbiotica strains. This tree supported the tree derived from the rpoB sequences and the multi-gene tree of

Ferreira et al. (2014). If the first portion of the gene was used, P. t. cinerea converged with the other P. temperata as in Figures 2-3 and 2-5 and presented by Tóth & Lakatos (2008). The problem with removing the potential recombinant portion in the P. t. cinera strains is that it allowed the recombinant portion in P. l. kleinii to be dominant.

The strain Pjun was identical to P. temperata subsp. cinerea strains pur1, pur2 and pur3

(KU559323-KU559325) and the 838 base overlap was identical to P. asymbitotica Ps

(KT899923). These strains most likely belong to the same species. It was suggested that P. t.

cinerea is more closely related to P. heterorhabditis than to P. temperata (Ferreira et al., 2014), which is supported by this study. This is further supported by the rep-PCR genomic profile, which linked strain Pjun with P. heterorhabditis. Depending on the region of the rpoB gene analyzed for the construction of phylogenetic trees P. t. cinerea was more closely related to P. heterorhabditis or to P. a. asymbiotica.

In the rpoB gene trees, P. a. australis is located among the P. luminescens strains, forming a monophyletic group with P. l. akhurstii. When a 598-nucleotide region (positions

379-976) was analyzed, P. a. australis formed a monophyletic group with P. a. asymbiotica and a larger clade with P. heterorhabditis and P. t. cinerea. The tree topology was discordant with other Photorhabdus phylogenies in that P. temperata and P. luminescens were more closely related, while the monophyletic group consisting of P. asymbiotica, P. heterorhabditis and P. t. cinerea was more distant. The rpoB trees that retained greater concordance with other phylogenies consistently positioned P. a. australis in the P. luminescens clade.

Recently, it was shown that H. downesi isolates found in close vicinity to one another were associated with two different bacteria, P. t. temperata and P. t. cinerea, (Maher et al.,

2017). The symbiotic P. t. cinerea was able to improve nematode survival under desiccating conditions, and the nematode was readily able to exchange bacterial symbionts (Maher et al.,

2017). This exchange of bacterial strains within the nematode establishes condtions under which lateral gene transfer could be facilitated.

The bacterial symbiont most often found associated with H. indica is P. l. akhurstii, but it has also been shown that the symbiotic bacteria associated with two strains of H. indica isolated in Japan represented a novel strain of P. asymbiotica (Kuwata et al., 2008). The nematode host for P. asymbiotica Kingscliff strain is H. gerradii, a nematode that is similar to H. indica

(Gerrard et al., 2006; Plichta et al., 2009). It would be plausible for a bacterial symbiont to switch nematode hosts, especially if the nematode is very similar to its natural host. Perhaps there has been an incident of host switching in H. indica, where one symbiont is able to improve the nematode fitness under a different condition. Recombination could play a role in the evolution of the bacterium that allows it to outcompete other bacteria for a nematode host.

In conclusion, this is the first report of P. heterorhabditis associated with H. zealandica in Florida and South Carolina. All other bacteria isolated from the nematodes in Florida represent strains of P. l. akhurstii and P. l. luminescens. These were quickly and easily identified using rep-PCR, which proved to be a useful tool for the identification of closely related strains.

More reference strains should be incorporated to look at the relationships among other subspecies. The different tree topologies presented here indicate tenuous phylogenetic inferences. The results of the recombination analyses performed with RDP4 suggested multiple events involving different taxa making it difficult, if not impossible, to eliminate all potential intragenic events. Because of the discordance of gene topologies derived from not only different housekeeping genes but from different regions of the genes, it is imperative to use multiple gene sequences to improve the likelihood of obtaining the correct tree. Even if regions chosen were free of intragenic events, analyses of the concatenated genes pointed to intergenic recombination.

With multiple genes there is a greater chance of overriding the phylogenetic signal from recently acquired gene fragments. With this in mind, more gene sequences should be investigated to work in combination with rpoB to infer evolutionary history among strains of Photorhabdus.

Table 2-1. Photorhabdus strains and form variants used for this study Strain Host Geographical origin Reference (source) P. asymbiotica subsp. asymbiotica 1216-79 Clinical specimen: Pennsylvania, USA Farmer et al., 1989 (= ATCC 43948) blood, 72-year-old (D. Bowen)a female 2407-88 Clinical specimen: Texas, USA Farmer et al., 1989 (= ATCC 43952) abdomen, (D. Bowen)a submandible, 36- year-old female 2617-87 Clinical specimen: Texas, USA Farmer et al., 1989 (= ATCC 43951) leg wound, 45-year- (D. Bowen)a old male 3105-77 Clinical specimen: Maryland, USA Farmer et al., 1989 (= ATCC 43949) blood, 80-year-old (D. Bowen)a female 3265-86T Clinical specimen: Texas, USA Farmer et al., 1989 (= ATCC 43950) pretibial wound, 78- (ATCC)2 3265-86T-2 (non- year-old male pigmented form)b P. heterorhabditis DE H. zealandica Florida, USA (K. Nguyen)c ENY H. zealandica Florida, USA (K. Nguyen)c F14 H. zealandica Florida, USA (K. Nguyen)3 FL480 H. zealandica Florida, USA This Study NCZ H. zealandica South Carolina, USA (K. Nguyen)c ZM H. zealandica South Carolina, USA (K. Nguyen)c P. luminescens subsp. luminescens ENY25 H. floridensis Florida, USA (K. Nguyen)c HbT (= ATCC H. bacteriophora Australia Poinar et al., 1977 29999, Brecon (R. Akhurst)a DSM 3368) FL332 H. floridensis Florida, USA Nguyen et al., 2006 ; Shapiro et al., 2014 (K. Nguyen)c MX4A H. mexicana Mexico Nguyen et al., 2004 (K. Nguyen)c MX4C H. mexicana Mexico (K. Nguyen)c SC4 Heterorhabditis sp. South Carolina, USA (K. Nguyen)c P. luminescens subsp. akhurstii FL237 H. indica Florida, USA This Study FL265 H. indica Florida, USA This Study FL301 H. indica Florida, USA This Study FL304 H. indica Florida, USA This Study FL319 H. indica Florida, USA This Study FL354 H. indica Florida, USA This Study

Table 2-1. Continued Strain Host Geographical origin Reference (source) P. luminescens subsp. akhurstii (continued) FL370 H. indica Florida, USA This Study FL376 H. indica Florida, USA This Study FL406 H. indica Florida, USA This Study FL479 H. indica Florida, USA This Study FL482 H. indica Florida, USA This Study FL498 H. indica Florida, USA This Study FL506 H. indica Florida, USA This Study HG1 H. indicad Georgia, USA Gardener et al., 1994 (K. Nguyen)c HG2 H. indicad Georgia, USA (K. Nguyen)c HSA15 H. indica Saudi Arabia (K. Nguyen)c HSA17 H. indica Saudi Arabia (K. Nguyen)c IND H. indica India Poinar et al., 1992 (K. Nguyen)c P. luminescens subsp. laumondii Arg H. bacteriophorae Argentina (K. Nguyen)c ArgB H. bacteriophorae Argentina (B. Adams)c HP88 H. bacteriophora Utah, USA Akhurst et al., 1996 HP88 (D. Bowen)a HP88-2 (non- pigmented form) HP88A H. bacteriophora Utah, USA (K. Nguyen)c HP88A-2 (non- HP88 pigmented form)b HP88B H. bacteriophora Utah, USA (B. Adams)c HP88 P. luminescens NC H. bacteriophora North Carolina, USA (K. Nguyen)c SC1 H. bacteriophora South Carolina, USA (K. Nguyen)c SC2 H. bacteriophora South Carolina, USA (K. Nguyen)c SC3 H. bacteriophora South Carolina, USA (K. Nguyen)c SC5 H. bacteriophora South Carolina, USA (K. Nguyen)c SC6 H. bacteriophora South Carolina, USA (K. Nguyen)c SC7 H. bacteriophora South Carolina, USA (K. Nguyen)c P. temperata subsp. temperata K122 H. downesi K122 Ireland Nielsen & Lübeck, 2002 (D. Bowen)a K122 GI H. downesi K122 Ireland (K. Nguyen)c Meg1 H. megidis England (K. Nguyen)c XlNachT H. megidis Russia Akhurst, 1987 (= DSM 14550) (R. Akhurst)a XlNachTB H. megidis Russia (S. Forst)a

Table 2-1. Continued Strain Host Geographical origin Reference (source) P. temperata subsp. cinerea Pjun H. megidis The Netherlands Gerritsen et al., 2005; Tóth & Lakatos 2008 (D. Bowen)a P. temperata subsp. khanii =P. temperata subsp. stackebrandtii NC19 (= ATCC H. bacteriophora North Carolina, USA Akhurst, 1983; 29304, NC1 Tailliez et al., 2010 DSM 3369) (D. Bowen)a NC19-2 (non- pigmented form)b P. temperata subsp. tasmaniensis MegDB H. megidis ? Poinar et al., 1987; Tailliez et al., 2010 (D. Bowen)a P. temperata Mar1 Heterorhabditis sp. USA K. Nguyen)c aProvided bacteria bSpontaneous change to non-pigmented form, not tested for 2o form characteristics. cProvided host nematode dsyn. H. hawaiiensis; esyn. H. argentinensis;

Table 2-2. DNA primers used for rep-PCR Repetitive Primer Oligonucleotide Sequence References Element BOX BOXA1R 5’-CTACGGCAAGGCGACGCTGACG-3’ Martin et al., 1992; Versalovic et al., 1994 ERIC ERIC1R 5’-ATGTAAGCTCCTGGGGATTCAC-3’ Versalovic et al., 1991 ERIC2 5’-AAGTAAGTGACTGGGGTGAGCG-3’ REP REP1R 5’-IIIICGICGICATCIGGC-3’ Versalovic et al., 1991 REP2I 5’-ICGICTTATCIGGCCTAC-3’

Table 2-3. Strains and GenBank accession numbers used for phylogenetic analyses Strain Host Geographical gyrB GenBank origin accession no. P. asymbiotica subsp. asymbiotica 3105-77 (ATCC43949) Clinical specimen Maryland, USA FM16259a P. asymbiotica subsp. australis 9802892 (DSM 17609) Clinical specimen Australia JONO01000012b GCH001 Clinical specimen Australia AY278500 MB Clinical specimen Australia AY278511 P. heterorhabditis Q614 Heterorhabditis sp Australia AY278514 VMG H. zealandica South Africa LJCS01000024c P. luminescens subsp. akhurstii D1 H. indica Australia AY278499 Tetuan Heterorhabditis sp Cuba AY278515 P. luminescens subsp. caribbeanensis HG29 (DSM 22391) H. bacteriophora Guadeloupe EU930360 P. luminescens subsp. hainanensis C8404 (DSM 22397) Heterorhabditis sp China AY278498 P. luminescens subsp. kayaii C8406 Heterorhabditis sp China AY322432 P. luminescens subsp. kleinii KMD37 (DSM 23513) H. georgiana Ohio, USA JX513407 P. luminescens subsp. laumondii HP88 H. bacteriophora Utah, USA AY278508 K80 Heterorhabditis sp Argentina AY278509 TT01 H. bacteriophora Trinidad BX571859d P. luminescens subsp. luminescens Hb H. bacteriophora Australia AY278501 MX4 H. mexicana Mexico FJ874737 P. luminescens subsp. noenieputensis AM7 Heterorhabditis sp South Africa JQ424884 P. luminescens subsp. “sonorensis” Caborca H. sonorensis USA JQ912647 CH35 H. sonorensis USA JQ912652 P. temperata subsp. cinerea 3107 H. downesi Hungary EU053168 P. temperata subsp. khanii Meg H. megidis USA AY278512 P. temperata subsp. stackebrandtii GPS11 H. bacteriophora Ohio GU249303 P. temperata subsp. tasmaniensis NZH3 H. zealandica New Zealand AY278513 T327 H. zealandica Australia EU930356 P. temperata subsp. temperata BE09 H. megidis ? EU930354

Table 2-3. Continued Strain Host Geographical gyrB GenBank origin accession no. P. temperata subsp. temperata H295 ? ? EF029050 K122 H. megidis Ireland EU930355 XlNach H. megidis Russia AY278517 P. temperata subsp. thracensis DSM15199 Heterorhabditis sp. Turkey CP011104e FR32 Not yet defined France EU930352 aP. asymbiotica subsp. asymbiotica complete genome, same accession number for rpoB gene sequence bP. asymbiotica subsp. australis rpoB located on different contig, rpoB accession number JONO01000037 cP. heterorhabditis rpoB located on different contig, rpoB accession number LJCS01000018 dP. luminescens subsp. laumondii complete genome, same accession number for rpoB gene sequence eP. luminescens subsp. thracensis complete genome, same accession number for rpoB gene sequence

Table 2-4. Models of nucleotide substitutions selected for gyrB and rpoB datasets Dataset Model Likelihood Parameters Substitution rate Nucleotide Proportion Γ matrix frequency of invariant distribution sites gyrB 48 taxaa 012234+I+G Ra =1.4900 Equal 0.3690 0.6210 Rb = 5.1184 Rc = 0.6409 Rd = 0.6409 Re =10.6135 Rf = 1.0000 gyrB 48 taxab GTR+I+G Ra = 1.2854 freqA = 0.2617 0.3610 0.6040 Rb = 4.4403 freqC = 0.2472 Rc = 0.6630 freqG = 0.2554 Rd = 0.4310 freqT = 0.2357 Re = 0.0751 Rf = 1.0000 gyrB 23 taxab SYM+I+G Ra = 1.5210 Equal 0.3460 0.5700 Rb = 5.0735 Rc = 0.6941 Rd = 0.4241 Re = 11.0712 Rf = 1.0000 rpoB 23 taxaa GTR+I+G Ra = 0.7643 freqA = 0.2366 0.490 0.5240 Rb = 4.497 freqC = 0.2236 Rc = 0.7930 freqG = 0.2662 Rd = 0.2129 freqT = 0.2736 Re = 10.3457 Rf =1.0000 rpoB 23 taxab GTR+I+G Ra = 0.8448 freqA = 0.2323 0.4840 0.5240 Rb = 4.9658 freqC = 0.2270 Rc = 0.9770 freqG = 0.2623 Rd = 0.2589 freqT = 0.2784 Re = 10.9114 Rf =1.0000 aFor maximum likelihood analysis bFor Bayesian analysis

Table 2-5. Pairwise homoplasy index test for recombination a Data set No. of Taxa No. of Mean Φw P-value informative sites All strains gyrB 47 214 0.41244 3.63E-10 rpoB 25 223 0.29536 2.584E-6 gyrB-rpoB 23 399 0.32183 0.0 concatenated P. luminescens gyrB 24 107 0.24599 0.1074 rpoBb 12 107 0.16822 0.2623 gyrB-rpoB 12 178 0.1262 0.00603 concatenated P. asymbiotica and P. heterorhabditis gyrB 9 83 0.14252 1.598E-7 rpoB 7 76 0.11123 0.4479 gyrB-rpoB 6 137 0.09049 0.1183 concatenated P. temperata gyrB 15 111 0.98935 0.0253 rpoB 6 48 0.0 1.0 gyrB-rpoB 5 80 0.07247 0.05784 concatenated aLength of data sets for gyrB, rpoB and gyrB-rpoB concatenated were 918, 1447 and 2365 nucleotides, respectively. bTest conducted without P. asymbiotica australis; however, no evidence of recombination with addition of P. a. australis to P. luminescens data set.

Table 2-6. Intragenic recombination events as detected using GARD Gene fragment Putative Model averaged LHS RHS breakpointa support P-valueb P-valuec gyrBd 490 0.988621 0.0002 0.0002 499 0.81976 0.0012 0.0002 rpoBd 724 0.361972 0.0002 0.033 rpoBe 328 0.210218 0.0004 0.0724 730 0.867508 0.006 0.002 aNumbers indicate the nucleotide number within the partial sequences (i.e. nucleotide number 490 of 918 nucleotides in gyrB). bAdjusted KH P-value indicating the topology inferred from the data to the left of the breakpoint is significantly different to that inferred by the data to the right. cAdjusted KH P-value indicating the topology inferred from the data to the right of the breakpoint is significantly different to that inferred by the data to the left. dSingle breakpoint. eTwo breakpoints.

Table 2-7. Putative recombination events in gyrB, rpoB and gyrB-rpoB concatenated sequences detected by RDP4 Recombinant Breakpoint Detection method sequence positionsa RDP GENECONV Bootscan Maxchi Chimaera Sciscan P-Value gyrB P. l. kleinii DSM23513 570 834 <0.001 <0.01 NS <0.001 <0.001 <0.001 568 865 <0.001 <0.01 <0.01 <0.001 <0.001 <0.001 P. t. cinerea Pjun, 23 534 <0.01 NS NS <0.001 <0.001 <0.001 P. t. cinerea 3107 534 911 <0.01 NS NS <0.001 NS <0.001 P. heterorhabditis Q614 764 878 <0.001 <0.01 NS NS NS <0.01 rpoB bP. t. cinerea Pjun 148 824 NS NS NS <0.001 <0.01 <0.001 b, cP. l. laumondii HP88, 640 1414 NS NS NS <0.01 NS <0.001 TT01 gyrB-rpoB P. t. cinerea Pjun 534 2332 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 b, cP. l. akhurstii FL406, 1552 1597 NS <0.001 NS NS NS <0.001 FL265 P. a. australis 160 867 NS NS NS <0.01 <0.01 <0.001 bP. heterorhabditis VMG, 922 1636 <0.01 NS NS <0.01 <0.01 <0.001 FL480 aNumbers indicate the nucleotide number within the partial sequences (i.e. nucleotide number 570 of 918 nucleotides in gyrB). bRecombinant sequence may have been misidentified. cPossible this event has been caused by an evolutionary event other than recombination.

Table 2-8. Pairwise homoplasy index test for recombination of portions of gene sequences suspected of recombination events Region of partial gene Sequence length P-value sequence gyrB 1-490 490 0.156* 491-918 428 0.003 1-499 499 0.138* 500-918 419 0.003 1-22, 535-918 406 0.009 23-534 512 0.195* 1-763, 879-918 803 1.548E-7 1-569, 835-918 655 4.158E-4 1-567, 866-918 620 5.108E-4 568-865 298 0.002 rpoB 1-147, 825-1447 770 0.009 148-824 677 0.057* 1-640, 1414-1447 674 0.034 641-1413 773 0.002 1-724 724 0.033 725-1447 723 0.006 1-328 328 0.366* 1-730 730 0.027 329-730 402 0.103* 731-1447 717 0.003 379-976 598 0.072* 1-378, 977-1447 849 0.006 *Region of gene where recombination is not detected.

Figure 2-1. Sampling sites containing entomopathogenic nematodes from which bacteria were isolated for this study. All sites contained Heterorhabditis spp. except for sample sites 224 and 446, which contained Steinernema glaseri. Sample site 332 contained H. floridensis and sample site 480 contained H. zealandica. All other sites contained H. indica. Soil was collected in areas adjacent to productive citrus groves. (Map created using GPS Visualizer, www.gpsvisualizer.com.)

20 40 60 80 100 BOX REP ERIC 2617-87 2407-88 P. asymbiotica

3265-86T asymbiotica 3265-86T-2 1216-79 3105-77 MAR1 P. temperata HP88A-2 HP88A HP88-2 HP88B HP88 ARG ARGB P. luminescens SC3 laumondii SC5 SC7 SC1 NC SC2 SC6 NC19 P. temperata NC19-2 khanii Pjun P. temperata F14 cinera ENY P. NCZ heterorhabditis DE ZM FL480 MEGDB XlNachT XlNach-B P. temperata MEG1 K122 GI temperata K122 FL332 ENY 25 SC4 P. luminescens MX4A luminescens MX4C FL479 FL354 A HSA15 HSA17 FL265 FL237 HG FL376 FL506 P. luminescens FL498 akhurstii FL482 HGB FL370 B FL319 FL301 FL304 C IND FL406 Figure 2-2. Cluster analysis of banding patterns of Photorhabdus strains. Genomic fingerprints were generated by rep-PCR using primers corresponding to BOX1A, REP and ERIC sequences. The dendrogram was constructed with UPGMA with similarity levels expressed as the percentage values of the Pearson product-moment correlation coefficient (r). Colored boxes indicate strains isolated for this study.

A 10

00 00

00 00 00 00 00 00 00

5 3 4 6 7 8 9

11 12 10 B 12

00 00 00 00 00 00

00 00 00 00 00 00 00 00

19 20 21 22 23 24

13 1000 11 12 14 15 16 17 18

Figure 2-3. Nucleotide variability of gyrB and rpoB gene sequences of Photorhabdus strains. A) gyrB gene sequences (positions 334-1250, E. coli numbering). B) rpoB gene sequences (positions 1000-2439). The x-axis represents nucleotide positions, and the y-axis indicates the mean variability per window of 50 nucleotides. The graph was constructed using SVARAP v2.1.2 software (Colson, et al., 2006).

P. luminescens luminescens Hb (*)/98 P. luminescens luminescens SC4 77 * (*) P. luminescens luminescens FL332 * /94 P. luminescens luminescens MX4 (89)/94 P. luminescens luminescens MX4A (*)/* P. luminescens ‘sonorensis’ Caborca * P. luminescens ‘sonorensis’ CH35 82 P. luminescens noenieputensis AM7 P. luminescens laumondii K80 P. luminescens laumondii TTO1 (*)/* P. luminescens laumondii HP88 82 * P. luminescens laumondii AY278505 P. luminescens kleinii DSM23513 P. luminescens kayaii C8406 * P. luminescens kayaii CIP108428 P. luminescens akhurstii FL265 P. luminescens akhurstii FL482 P. luminescens akhurstii FL370 P. luminescens akhurstii Tetuan P. luminescens akhurstii FL406 P. luminescens akhurstii D1 (86)/86 * P. luminescens akhurstii FL354 (*) /98 P. luminescens akhurstii IND * P. luminescens hainanensis C8404 P. luminescens caribbeanensis HG29 (96)/94 P. temperata khanii Meg (97)/90 * P. temperata khanii NC19 99 P. temperata stackebrandtii GPS11 99 P. temperata Mar1 94 P. temperata tasmaniensis Meg (*)/* P. temperata tasmaniensis NZH3 * * P. temperata tasmaniensis T327

P. temperata temperata Meg1

(88) /98 * P. temperata temperata BE09 (88) /88 93 P. temperata temperata XlNach

(99)/96 * P. temperata temperata K122

* P. temperata thracensis FR32 93 P. temperata thracensis DSM15199 P. temperata cinerea PJun (*) /76 * * P. temperata cinerea H3107 * P. asymbiotica austrailis MB (*)/* P. asymbiotica austrailis 9802892

* P. asymbiotica austrailis GCH001

88 * P. asymbiotica asymbiotica 3105-77 P. asymbiotica asymbiotica 3265-86 71 (*)/* P. heterorhabditis VMG (99) /* * P. heterorhabditis FL480 * P. heterorhabditis Q614 X. nematophila ATCC19061 (*) /98 E. coli CFT073 * Y. pestis CO92

Figure 2-4. Phylogenetic tree from ML analysis of gyrB partial gene sequences for 48 Photorhabdus strains constructed with GARLI. Support values greater than 70 are presented above and below branches. Numbers above branches represent values for 1000 bootsrap replicates for maximum parsimony and likelihood (MP)/ML. Numbers below branches represent posterior probability as percentages. All values =100 represented as *. Highlighted areas indicate positions of Florida isolates.

Figure 2-5. Phylogenetic relationships between species/strains of Photorhabdus based on maximum likelihood analysis of partial rpoB gene sequences constructed in GARLI. Support values greater than 70 are presented above and below branches. Numbers above branches represent values for 500 bootsrap replicates for maximum parsimony and likelihood (MP)/ML. Numbers below branches represent posterior probability as percentages, values =100 represented as *.

FL482 FL406 99 FL482 FL370 * * FL354 95 FL370 gyrB P. luminescens akhurstii FL406 rpoB FL265 * * IND IND 90 FL354 91 FL265 SC4 P. asymbiotica australis 95 Hb1 DSM17609 * FL 332 * * * FL332 P. luminescens luminescens SC4 * MX4A Hb1 * 98 TT01 * MX4A P. luminescens laumondii HP 88 HP88 * ATCC43949 TT01 * P. asymbiotica asymbiotica 99 ATCC43950 ATCC43950 * P. asymbiotica australis 95 83 ATCC43949 DSM17609 * * FL480 FL 480 P. heterorhabditis 99 VMG VMG * * K122 * P. temperata cinerea Pjun Xl Nach 91 K122 P. temperata temperata P. temperata Mar1 Xl Nach * 87 P. temperata khanii NC19 P. temperata Ma r1 87 P. temperata thracensis * P. tempe rata khanii NC19 * DSM 15199 P. temperata cinerea Pjun P. temperata thracensis DSM 15199

Figure 2-6. Comparsion between the gyrB gene (on left) and rpoB gene (on right) Bayesian trees. Trees were constructed using MrBayes. Posterior probability as percentages located above branches, values =100 represented as *. Because the rpoB sequence is longer than the gyrB sequence by more than 500 nucleotides, the rpoB gene sequence was reduced to 1038 nucleotides for this anlalysis. Trees were rooted with X. nematophila ATCC 19061, E. coli strain CFT073 and Y. enterocolitica strain 8081.

91 P. luminescens akhurstii FL482 P. luminescens akhurstii FL370

P. luminescens akhurstii FL354 P. luminescens akhurstii FL406 100 P. luminescens akhurstii FL265

P. luminescens akhurstii IND

99 P. luminescens luminescens FL332

100 P. luminescens luminescens SC4 99 100 P. luminescens luminescens Hb

P. luminescens luminescens MX4A

100 P. luminescens laumondii TTO1 81 P. luminescens laumondii HP88

100 P. asymbiotica asymbiotica 3105-77 81 P. asymbiotica asymbiotica 3265-86 P. asymbiotica austrailis 9802892 84 100 P. heterorhabditis FL480 P. heterorhabditis VMG

P. temperata cinerea PJun 99 100 P. temperata Mar1 85 P. temperata khanii NC19

100 P. temperata temperata K122 83 100 P. temperata temperata XlNach

P. temperata thracensis DSM15199

X. nematophila ATCC19061

Y. pestis CO92

E. coli CFT073 0.05 Figure 2-7. Phylogenetic relationships between species/strains of Photorhabdus based on maximum likelihood analysis of concatenated gyrB-rpoB partial gene sequences constructed in GARLI. Model of nucleotide substitution for gyrB (012232+I+G) and for rpoB (TIM3+I+G). Numbers above branches represent values for 300 bootsrap replicates. Support values greater than 70 are presented.

A 0.01 P. l. luminescens MX4 P. luminescens P. l. "sonorensis" CH35 MX4A P. l. "sonorensis" Caborca

P. l. luminescens Hb, FL332, SC4 P. l. caribbeanensis HG29 P. l. noenieputensis AM7

P. l. hainanensis C8404 P. l. laumondii HP88 P. l. laumondii K80 SC3

P. l. akhurstii IND P. l. kayaii ITH-LA3 P. l. kayaii FRM17 P. l. akhurstii Tetuan FL354 FL406 P. l. akhurstii D1, FL265 P. l. kayaii FR33

P. l. kleinii DSM23513 P. l. kleinii OH25

B P. a. asymbiotica 3268-86, 3105-77 0.01 P. asymbiotica

P. a. australis 9802892

P. sp. Q614 P. heterorhabditis FL480 P. t. cinerea Pjun

P. temperata

P. t. thracensis 36-8, H3210

P. t. khanii Meg P. t. khanii NC19

P. t. stackebrandtii GPS11, Mar1

P. t. temperata XlNachB P. t. temperata K122 P. t. temperata XlNach P. t. tasmaniensis NZH3, T327, MEG WI Figure 2-8. Split decomposition graphs. A) P. luminescens strains. B) P. asymbiotica, P. heterorhabditis and P. temperata strains. The networks were constructed using gyrB partial DNA sequences using the uncorrected P characters, fit scores are 62.85 for A and 64.30 for B.

0.01 P. a. asymbiotica 3268-86, 3105-77 P. asymbiotica P. a. australis 9802892

P. t. cinerea Pjun FL482 FL370 FL406 FL354 P. l. akhurstii IND FL480 FL265 VMG P. heterorhabditis

P. luminescens

SC4 FL332 P. l. luminescens Hb P. temperata P. l. luminescens MX4A Hp88 P. l. laumondii TT01 P. t. khanii NC19 Mar1

P. t. temperata XlNach, K122

P. t. thracensis DSM15199 Figure 2-9. Split decomposition graph for Photorhabdus strains. The graph was constructed using rpoB partial DNA sequences using the uncorrected P characters transformation. The scale bar represents the number of substitutions per site. The fit score is 63.997.

0.01 P. luminescens P.l. akhurstii IND, FL265, FL406 P. l. akhurstii FL482, FL370 P. l. laumondii TT01 P. l. laumondii HP88 P. l. akhurstii FL354

P. a. austrailis 9802892

P. asymbiotica FL332 SC4 P. l. luminescens Hb1 P. a. asymbiotica 3268-86 P. a. asymbiotica 3105-77 P. l. luminescens MX4A

P. heterorhabditis VMG FL480 P. heterorhabditis

P. temperata P. t. cinerea Pjun

P. t. khanii NC19 Mar1 P. t. thracensis DSM15199 P. t. temperata K122 P. t. temperata XlNach

Figure 2-10. Split decomposition graph for Photorhabdus strains. The network was constructed using concatenated gyrB-rpoB partial DNA sequences using the uncorrected P characters transformation. The scale bar represents the number of substitutions per site. The fit score is 72.956.

FL406 FL406 Tetuan Tetuan 95 B A 97 FL482 FL482 FL370 FL370 FL265 FL265 D1 FL354 * Recombinant * 77 IND D1 * Minor Parent FL354 IND * 77 P

C8404 Major Parent C8404 l

HG29 HG29 m

C8406 Caborca i * n Caborca CH35 e * SC4 s CH35 c

99 * MX4 FL332 * e * * n MX4A Hb * 73 s

* 95 SC4 MX4 99 Hb MX4A * * FL332 AM7 AM7 K80 HP88 TT01 K80 HP88 * 97 * TT01 HP88FL HP88FL P. l. kleinii DSM23513 * * 3265-86 C8406 3105-77 9802892 98 9802892 P. asymbiotica MB * * * MB GCH001 91 GCH001 3265-86 * FL480 3105-77 98 * VMG P. heterorhabditis FL480 94 VMG * Q614 * * * PJun P. t. cinerea Q614 * 3107 NZH3 94 NZH3 Meg 99 * T327 T327

Meg DB P. temperata Mar1 * P 84 98 . 71 93 P. temperata Mar1 DSM23271

t 96 DSM23271 Meg 98 e Meg NC19 m 92 p

H3210 97 e 76 NC19

H3210 DSM15199 96 a

* Pjun t DSM15199 P. t. cinerea a

* 94 K122 3107 XlNach K122 * H295 XlNach * Meg 1 H295 P. l. kleinii DSM23513 Meg 1 91 Figure 2-11. Alternative tree topologies given by different regions of the gyrB partial gene sequence (918 bp). Breakpoints determined using RDP4. Bayesian trees constructed using MrBayes 3.2.6. A) With nucleotides 1-22 and 535-918 (406 bp). B) With nucleotides 23-534 (512 bp). Posterior probability as percentages 70 or greater located above branches, values =100 represented as *. Trees were rooted with X. nematophila ATCC 19061, E. coli strain CFT073 and Y. pestis CO92. Both trees were constructed using the SYM + I + Γ model of nucleotide evolution.

1-569, 835-918

Figure 2-12. Position of P. heterorhabditis Q614, P. temperata subsp. cinerea and P. luminescens subsp. kleinii DSM23513 as determined by region of the gyrB gene used for phylogenetic reconstruction. A) Breakpoint of 490 determined using GARD. Different sides of the breakpoint yield trees with different positions for P. heterorhabditis Q614 and P. temperata subsp. cinerea. B) Tree showing the position of P. luminescens subsp. kleinii DSM23513 once putative recombinant portion is removed. Breakpoints of 570 and 834 determined using RDP4. Bayesian trees constructed using MrBayes 3.2.6. Posterior probability as percentages 70 or greater located above branches, values =100 represented as *. Trees were rooted with X. nematophila ATCC 19061, E. coli strain CFT073 and Y. pestis CO92. Both trees were constructed using the SYM + I + Γ model of nucleotide evolution.

FL304 FL304 78 * FL319 FL319 A 74 B FL482 FL482 88 70 FL370 FL370 98 Recombinant FL406 P. l. akhurstii FL354 Minor Parent 78 FL354 FL265 71 * Major Parent * FL265 FL406 IND IND * LN2 LN2 P. a. austrailis 9802892 FL332 * * FL332 SC4 * 87 * SC4 P. l. luminescens Hb 99 * * Hb MX4A MX4A P. l. laumondii HP88 * * P. l. laumondii HP88 P. l. laumondii TT01 P. l. laumondii TT01 P. t. temperata K122 97 93 FL480 P. t. temperata XlNach * P. heterorhabditis 78 73 DE P. t. temperata Meg1 P. t. cinerea PJun P. temperata thracensis * DSM15199 * 98 VMG P. t. khanii Mar1 * * * * 3105-77 P. t. khanii NC19 3265-86 FL480 P. t. temperata K122 VMG 98 * P. t. temperata XlNach DE * P. t. temperata Meg1 P. t. cinerea PJun 98 P. t. khanii Mar1 3265-86 * P. a. asymbiotica * P. t. khanii NC19 3105-77 99 P. temperata thracensis P. a. austrailis 9802892 DSM15199 Figure 2-13. Alternative tree topologies based upon different regions of the rpoB gene. Breakpoints determined using RDP4, and Bayesian trees constructed using MrBayes 3.2.6. Tree A constructed with nucleotides 1-378 and 977-1447 (849 nt) using the GTR + I + Γ model of nucleotide evolution, and tree B constructed with nucleotides 379-976 (598 nt) using the SYM + I + Γ model of nucleotide evolution. Posterior probability as percentages 70 or greater located above branches, values =100 represented as *. Trees were rooted with X. nematophila ATCC 19061, E. coli strain CFT073 and Y. pestis CO92.

FL304 FL304 97 FL319 FL319 * A 98 B FL482 FL482 95 99 FL370 FL354 88 Recombinant FL406 P. l. akhurstii FL370 Minor Parent * FL354 FL265 82 Major Parent * FL265 FL406 * IND IND 94 70 LN2 LN2 95 P. a. austrailis 9802892 FL332 * * 99 FL332 SC4 * * SC4 P. l. luminescens Hb 99 * * Hb MX4A 88 MX4A P. l. laumondii HP88 * P. l. laumondii HP88 P. l. laumondii TT01 * P. l. laumondii TT01 P. a. austrailis 9802892 92 * FL480 FL480 DE DE 96 P. heterorhabditis * PJun VMG 94 96 VMG 3265-86 91 * 3265-86 P. a. asymbiotica 3105-77 * * 3105-77 PJun 91 P. t. temperata K122 P. t. khanii Mar1 * * P. t. temperata XlNach P. t. khanii NC19 P. t. temperata Meg1 P. t. temperata K122 * * P. t. khanii Mar1 P. t. temperata XlNach * * 88 P. t. khanii NC19 P. t. temperata Meg1 P. temperata thracensis P. temperata thracensis DSM15199 DSM15199 Figure 2-14. Alternative tree topologies based upon different regions of the rpoB gene. Breakpoints determined using RDP4, and Bayesian trees constructed using MrBayes 3.2.6. Tree A constructed with nucleotides 1-147 and 825-1447 (770 nt) and tree B constructed with nucleotides 148-824 (677 nt). Both trees constructed using the SYM + I + Γ model of nucleotide evolution. Posterior probability as percentages 70 or greater located above branches, values =100 represented as *. Trees were rooted with X. nematophila ATCC 19061, E. coli strain CFT073 and Y. pestis CO92.

Figure 2-15. Maximum likelihood tree of concatenated gyrB-rpoB sequences with potential regions of recombination removed. Nucelotide 23-534 of 918 for gyrB and nucleotides 379-976 of 1447 for rpoB were used for analyses. ML boostrap support values 70 or greater above branches. Posterior probability as percentages 70 or greater located above branches, values =100 represented as *. Trees were rooted with E. coli strain CFT073 and Y. pestis CO92.

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CHAPTER 3 GENETIC DIVERSITY AMONG Xenorhabdus SPP. AND THEIR PHYLOGENETIC INFERENCE

Introduction

Nematodes of the family Steinernematidae are obligate parasites of insects and are characterized by having a mutualistic association with bacteria of the genus Xenorhabdus

Thomas and Poinar, 1979. The third stage infective juvenile (IJ), the only free-living form of the nematode, harbors cells of its bacterial symbiont within the intestine. The bacterial cells are contained in a specialized structure located at the anterior end of the intestine called the bacterial receptacle (Kim et al., 2012), formerly known as the bacterial vesicle (Akhurst & Bird, 1983).

The bacteria are released following entry of the IJ into the hemocoel of the insect. Within the insect Xenorhabdus cells proliferate, killing the insect and bioconverting the tissue into nutrients that optimize nematode growth and reproduction (Forst & Clarke, 2002). Once high cell densities have been reached, the bacteria produce an array of antimicrobial compounds that protect the insect cadaver from colonization by other organisms (Akhurst, 1982; Akhurst &

Boemare, 1988; Boemare et al., 1997a; Webster et al., 2002). The nematodes produce up to three generations, and once the insect host resources have been consumed, new IJs, colonized by their Xenorhabdus symbiont, exit into the environment.

At least 95 species of steinernematid nematodes have been described (Hunt, 2016), each presumably having a specific mutualistic relationship with Xenorhabdus. Xenorhabdus are similar only to the bacterial symbiont of heterorhabditid nematodes, Photorhabdus. They are

Gram-negative, nonfermentive rod-shaped bacteria currently belonging to the family

Enterobacteriaceae (Thomas &Poinar, 1979). It recently was proposed to move Xenorhabdus to a new family Morgenallacea (Adeolu et al., 2016). At the time of this writing, 24 species of

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Xenorhabdus have been described. As new nematodes and accordingly bacterial species are being described, their evolutionary history and taxonomic diversity are continuing to emerge.

The most commonly used gene for evolutionary and systematic studies of prokaryotes is the16S (small subunit) rRNA. However, the small subunit ribosomal RNA has been documented to undergo lateral gene transfer and often provides insufficient data for the resolution of phylogenies due to the presence of both highly variable and highly conserved regions (Lerat et al., 2003, Naum et al., 2007). Protein coding genes have been used successfully to describe phylogenetic relationships. The single copy genes gyrB (encoding the ATPase domain of DNA gyrase) (Dauga, 2002; Akhurst et al., 2004; Gerrard et al., 2006) and recA (recombinase A, involved in DNA repair) (Sergeant et al., 2006) have been proposed as suitable genes for reconstruction of phylogenies of bacteria within the genus and genera closely related to

Xenorhabdus. Concatenated sequences of gyrB, recA, dnaN (beta subunit of DNA polymerase

III), gltX (glutamate tRNA ligase) and infB (translation initiation factor IF-2) have been used to examine relationships among Xenorhabdus and to describe new Xenorhabdus species (Tailiez et al., 2012; Ferreira et al., 2013a; Kuwata et al., 2013). Phylogenetic relationships among

Xenorhabdus have been described using recA and serC (phosphoserine aminotransferase) individually and combined (Lee & Stock, 2010a; 2010b). Two genes that have been successfully used for determination of bacterial phylogeny within the family Enterobacteriaceae are the - subunit of RNA polymerase (rpoB) (Mollet et al., 1997; Hoffmann & Rogenkamp, 2003;

Stephan et al., 2007; Behrendt et al., 2015) and the 60 kDA chaperonin, heat shock protein 60

(groEL) (Hoffmann & Rogenkamp, 2003; Hoffmann et al., 2005; Gillis et al., 2014). Like gyrB and recA, rpoB and groEL are single copy genes that are universally present in bacteria. The

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genes recA, rpoB and groEL are considered to be genes that have a low likelihood of undergoing lateral gene transfer (Lerat et al., 2003).

For this study the genetic diversity and evolutionary history of Xenorhabdus from several different steinernematid nematode isolates was investigated. Species of Xenorhabdus were selected on the basis of diversity according to profiles generated by rep-PCR. Their phylogenetic relationships were inferred based on two housekeeping genes from previous studies, gyrb and recA, and two housekeeping genes that have not been used for the study of relationships among Xenorhabdus, rpoB and groEL. An objective of this study was to demonstrate the utility of rpoB and groEL for the phylogeny of Xenorhabdus along with rep-

PCR, which could allow better understanding of the distribution and relationships among these symbionts. Because bacterial recombination can generate misleading evolutionary data, the possiblility of recombination was explored

Materials and Methods

Collection of Nematode Specimens, Isolation of Bacterial Strains and Growth Conditions

Bacterial symbionts were isolated from steinernematid nematodes generously provided by Dr. Larry Duncan (University of Florida, CREC, Lake Alfred), Dr. Harry Kaya (University of

California, Davis), Dr. Albrecht Koppenhöfer (Rutgers University, New Brunswick, NJ), Dr.

Khuong Nguyen (University of Florida, Gainesville), Dr. David Shapiro-Ilan (USDA-ARS,

Byron, GA) and Dr. Robin Stuart (University of Florida, CREC, Lake Alfred). The entomopathogenic nematodes were maintained in the Nematode Evolution Laboratory at the

University of Florida, Gainesville. A total of 17 known and two undescribed species within the genus Steinernema were included in this study. To isolate the symbiotic bacteria, infective juveniles (IJs) were surface sterilized by incubating 30 min in a solution of 0.125% (w/v) methylbenzathonium chloride with gentle rocking, followed by a 15 min incubation in 3% (v/v)

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hydrogen peroxide. The IJs were triple rinsed with sterile tap water and macerated to liberate their symbiotic bacteria. Xenorhabdus strains were identified based on morphological growth characteristics exhibited when cultured on Tergitol-7 Agar (BBL, Becton Dickinson and

Company, Franklin Lakes, NJ) supplemented with 0.004% triphenyltetrazolium chloride.

Additional bacteria were kindly provided by Dr. Ray Akhurst (CSIRO Entomology, Canberra

ACT 2601, Australia), Dr. David Bowen (Department of Animal Health and Biomedical

Sciences, University of Wisconsin, Madison), Dr. Steven Forst (Department of Bacteriology,

University of Wisconsin, Milwaukee) and Ms. Peggy Mandanas (Certis USA LLC, Columbia,

MD). All Xenorhabdus strains, representing 14 known and 3 undescribed species, were maintained on Luria-Bertani (LB) agar plates at 27ºC. Details of the origin and host affiliation of strains used in this study are presented in Table 3-1.

Preparation of Genomic DNA

Total genomic DNA was prepared by a modified method of Ausubel (1992). Briefly, cultures were grown overnight at 27°C in 5 ml of Luria-Bertani broth with shaking at 150 rpm.

Cells were collected by centrifugation of 1 ml of broth culture at 8,000 rpm for 3 min. The resultant pellet was washed with 500 µl sterile tap water, and the cells were again collected by centrifugation. The washed bacterial cells were incubated in 600 µl cell lysis buffer (10 mM

Tris-HCl, pH 8.0; 1 mM EDTA; 0.5% (w/v) sodium dodecyl sulfate; 30 µg proteinase K) for 1 h at 37°C. The nucleic acids were purified from the cell lysates by incubation with 150 µl of 5 M sodium chloride and 80 µl CTAB/NaCl solution (10% (w/v) cetyl trimethylammonium bromide in 0.7 M sodium chloride) for 10 min at 65°C followed by extractions with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1). Nucleic acids were precipitated with 0.6 vol isopropanol, washed twice with 70% (v/v) ethanol and

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dissolved in TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). The DNA was quantified spectrophotometrically.

Repetitive Sequence-based Genotyping

Genomic fingerprints were generated for Xenorhabdus strains using primers corresponding to the conserved bacterial repetitive elements BOX (BOXA1R, 5'-

CTACGGCAAGGCGACGCTGACG-3') (Martin et al., 1992; Versalovic et al., 1994), REP

(REP1R, 5'-IIIICGICGICATCIGGC-3' and REP2I, 5'-ICGICTTATCIGGCCTAC-3') and ERIC

(ERIC1R, 5'-ATGTAAGCTCCTGGGGATTCAC-3' and ERIC2, 5'-

AAGTAAGTGACTGGGGTGAGCG-3') (Versalovic et al., 1991). The rep-PCR was performed according to the methods described by Louws and Cuppels (2001) with slight modification.

Each reaction mixture contained 1 µl purified DNA (500 ng µl-1 for ERIC and REP-PCR or 50 ng µl-1 for BOX-PCR), 1 × Gitschier buffer (16.6 mM ammonium sulfate; 67 mM Tris HCl pH

8.8; 6.7 mM magnesium chloride; 6.7 µM EDTA pH8.8 and 30 mM ß-mercapto-ethanol), 10%

(v/v) dimethyl sulfoxide, 4.0 mg bovine serum albumin, 1.25 mM each dNTP, 0.3 µg of each primer (only one primer used for BOX-PCR), 2 U AmpliTaq DNA polymerase (Applied

Biosystems, Foster City, CA), and water to a final volume of 25 µl. The PCR amplifications were carried out in a PTC-100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) using the following thermal profile: initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 94°C for 3 s and 92°C for 30 s, annealing at 40°C (REP-PCR) or 50°C (BOX- and ERIC-PCR) for 1 min and extension at 65°C for 8 min with a final extension step at 65°C for

8 min. PCR products were subsequently held at 4°C until they were resolved by gel electrophoresis. A 12.5 µl aliquot of amplified PCR products was separated by gel electrophoresis on 1.5% (w/v) agarose gels in 1× Tris-acetate-EDTA buffer. The gels were stained with ethidium bromide and photographed on a UV transilluminator. A computer assisted

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pattern analysis was performed with the digital images using GelCompar software version 4.0

(Applied Maths, Sint-Martens-Latem, Belgium). The images were normalized using a 1 kb

DNA ladder (Invitrogen, Carlsbad, CA) loaded in the first and every ninth well of the agarose gel. Excessive background was subtracted by applying the rolling-disk option, and DNA bands detected by the software were confirmed by visual examination. The BOX, REP and ERIC generated fingerprints were linearly combined, and the similarity between pairs of the combined fingerprints was calculated using the product-moment correlation coefficient (r value) (Pearson,

1926) applied to the whole densiometric curve of the gel tracks (Rademaker et al., 2000; Louws

& Cuppels, 2001). A cluster analysis of the pairwise similarity values was performed using the unweighted pair group method using arithmetic averages clustering technique, and a dendrogram was constructed (Rademaker et al., 1999).

Gene Selection and Amplification

The housekeeping genes were selected based on the criteria that the genes (a) have no known paralogs, (b) are not in close proximity on the genome so as to not be co-transducible, (c) encode for proteins that have conserved functions, (d) are important for different physiological processes to reduce the chances of having coevolved and (e) have been used successfully in the construction of phylogenies for other genera within the family Enterobacteriaceae. Figure 3-1 is a diagram of the selected housekeeping genes and their respective map positions on the genome of X. nematophila ATCC19061. Inspection of the genomes for X. bovienii SS-2004, X. doucetiae strain FRM16 and X. poinarii strain G6 confirmed that the housekeeping genes are not in close proximity on these other Xenorhabdus chromosomes. Primers used for amplification and nucleotide sequencing are presented in Table 3-2. Regions of gene sequences used in this study are presented in Figure 3-2. All PCR was performed using a PTC-200 DNA Engine Peltier thermocycler (Bio-Rad Laboratories).

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The gyrB Sequence

The primers of Dauga (2002) were used to amplify a 971 nucleotide region of gyrB. The reaction mixture (25 l total vol) contained 1 × PCR buffer, 2.5 mM MgCl2, 0.2 µM each deoxynucleoside triphosphate, 1 µM each forward and reverse primer, 1 U Taq polymerase

(Promega Corporation, Madison, WI) and 1 l DNA template. The thermal cycling program consisted of an initial denaturation step for 3 min at 94C, 30 cycles of denaturation at 95C for

30 s, annealing at 52C for 1 min and extension at 72C for 2 min and a final extension step at

72C for 10 min.

The recA Sequence

Primers were designed based on the conserved regions between the sequences of X. bovienii (accession number U87924) and X. nematophila (AF127333). The forward primer annealed to the ribosomal binding region located just before the start codon and the reverse primer annealed to a conserved region within Xenorhabdus at nucleotide bases 840-861 (E. coli numbering). The reaction mixture (25 l total vol) contained 1 × PCR buffer, 2.5 mM MgCl2,

0.2 µM each deoxynucleoside triphosphate, 1 µM each forward and reverse primer, 1 U Taq polymerase (Promega Corporation) and 1 l DNA template. The thermal cycling program consisted of an initial denaturation step for 3 min at 95C, 30 cycles of denaturation at 95C for

30 s, annealing at 50C for 1 min and extension at 72C for 1.5 min and a final extension step at

72C for 10 min.

The groEL Sequence

Nearly the entire groEL sequence was amplified using the primers of Fares et al. (2002), which annealed to the groES gene and the terminal nucleotides of groEL. Each reaction mixture had a total volume of 50 l containing 1 × PCR buffer, 2.5 mM MgCl2, 0.2 µM each

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deoxynucleoside triphosphate, 1 µM each forward and reverse primer, 1 U Taq polymerase

(Promega Corporation) and 1 l DNA template. The thermal cycling program consisted of an initial denaturation step for 3 min at 95C, 30 cycles of denaturation at 95C for 30 s, annealing at 50C for 30s and extension at 72C for 2.5 min and a final extension step at 72C for 10 min.

The rpoB Sequence

For the amplification of rpoB, primers were designed on the basis of sequence conservation in Photorhabdus luminescens subsp. laumondii TT01 (accession number

BX571860), Escherichia coli O157:H7 EDL933 (AE005174), Salmonella enterica serovar

Typhi CT18 (AL627279), Shigella dysenteriae Sd197 (CP000034) and Shigella sonnei Ss046

(CP000038). The forward primer annealed at positions 974-978 and the reverse primer annealed at positions 2503-2525 allowing for the amplification sequence encoding part of the variable polypeptide region 3 and all of region 4 (Lisitsyn et al., 1988). Each reaction mixture had a total volume of 25 l containing 1 × PCR buffer, 2.5 mM MgCl2, 1 M betaine, 12.5 g bovine serum albumin, 0.2 µM each deoxynucleoside triphosphate, 0.5 µM each forward and reverse primer, 1

U Taq polymerase (Promega Corporation) and 0.5 l DNA template. The thermal cycling program consisted of an initial denaturation step for 3 min at 95C, 30 cycles of denaturation at

95C for 30 s, annealing at 53C for 1 min and extension at 72C for 2 min and a final extension step at 72C for 10 min.

The 16S rDNA Sequence

The relationships of the 16S rDNA sequences for two undescribed strains, Xenorhabdus sp. strain TX-26 and strain VN13, with known Xenorhabdus spp. were investigated. The primers of Ponsonnet & Nesme (1994) were used to amplify the 16S small subunit, the internal transcribed spacer region and part of the 23S large subunit. Each reaction mixture had a total

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volume of 50 l containing 1 × PCR buffer, 2.5 mM MgCl2, 0.2 µM each deoxynucleoside triphosphate, 1 µM each forward and reverse primer, 1 U Taq polymerase (Promega

Corporation) and 1 l DNA template. The thermal cycling program consisted of an initial denaturation step for 4 min at 95C, 30 cycles of denaturation at 94C for 30 s, annealing at 52C for 1 min and extension at 72C for 2.5 min and a final extension step at 72C for 10 min.

Cloning and Sequencing

For each DNA amplification, 5 l vol of PCR product was electrophoresed on 0.7%

(w/v) agarose containing ethidium bromide (10 mg ml-1) and visualized under UV light to ensure the amplicon was the correct size. The gyrB, recA groEL and rpoB amplicons were purified using the QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA). For 16S rDNA amplifications, 20 l of the amplified product was electrophoresed on a 5% (w/v) agarose gel, the DNA bands were excised and then purified using the Wizard Gel Purification® Kit (Promega

Corporation). Housekeeping genes were sequenced directly using the forward and reverse PCR primers. For sequencing of larger genes, groEL and rpoB, additional primers were designed as described above and used to sequence the internal portion of the gene products.

For 16S rDNA or housekeeping gene products that failed with direct sequencing, either the PCR products were ligated into a T-overhang vector using the pGEM®-T Vector System

(Promega Corporation), which was then transformed into MAX Efficiency® DH5α™

Competent Cells (Invitrogen), or the TOPO TA Cloning® kit (Invitrogen) was used according to the manufacturer's instruction. The presence of cloned inserts was verified by colony-PCR.

Each colony was picked with a sterile toothpick; part of the colony was transferred to the surface of an LB agar plate containing ampicillin and the remaining part placed into thin-walled tube containing 45 µl of sterile distilled water. The cells were lysed by boiling for 7 min and

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collected by centrifugation at 10,000 x g for 10 min. The supernatant (10 µl) was withdrawn and used for the template, and each reaction mixture (25 l total vol) contained 1 × PCR buffer, 1.5 mM MgCl2, 0.2 µM each deoxynucleoside triphosphate, 0.2 µM each primer (either T7 and Sp6 promoter primers or T7 promoter primer and reverse PCR primer) and 1 U Taq polymerase

(Promega Corporation). The thermal profile consisted of an initial denaturation of 95C for 3 min, followed by 35 cycles of denaturation at 94C for 1 min, annealing at 50C for 1 min, extension at 72C for 2 min and a final extension step at 72C for 10 min. Insert-containing colonies were grown overnight in LB broth containing ampicillin and purified using the QIAprep

Spin Miniprep Kit (Qiagen Inc.).

Purified gene products were sequenced using the ABI BigDye® Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) using a modified protocol. Each sequencing reaction contained 2.0 l BigDye® ready reaction mix, 2.0 l ICBR mix

(proprietary, University of Florida), 3.2 pM sequencing primer, 15 ng (PCR product) or 350 ng

(plasmid) template and purified water to a final reaction volume of 10 l. The following thermal profile was used for amplification: denaturation at 96C for 30 s, primer annealing at 50C for

15 s and product extension at 40C for 4 min for a total of 25 cycles and then held at 4 C. The sequencing products were purified by ethanol precipitation. Each sequencing reaction product was placed into a 1.5 ml microcentrifuge tube containing 30.0 l of cold 95% ethanol and 1.0 l

3 M sodium acetate, pH 5.2, incubated on ice for 10 min and centrifuged at 12,000 x g for 15 min. The ethanol mixture was removed, and the resulting pellet was rinsed with 250 l of 70% ethanol and dried using a rotary evaporator. Purified sequencing reaction products were recorded at the University of Florida ICBR DNA Sequencing Facility, Gainesville, FL with an

ABI 3130 automated DNA sequencer (Applied Biosystems).

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Alignments

Nucleotide sequences were assembled and edited using Sequencher software (Gene

Codes Corporation, Ann Arbor, Michigan). For all other taxa (Table 3-3) gyrB, recA, groEL and rpoB gene sequence data were obtained from GenBank (National Center for Biotechnology

Information, http://www.ncbi.nlm.nih.gov/genbank/). Nucleotide sequences were aligned with

Clustal X v2.1 (Larkin et al., 2007) using default parameters. The alignments were optimized manually using MacClade 4.06 (Maddison & Maddison, 2005) based on the translated amino acid states. The sequences were concatenated to form two combined molecular data sets: 1) consisting of two loci (gyrB (845 bp) and recA (646 bp); 1491 bp and 57 taxa) and 2) consisting of all 4 loci (groEL (1609 bp), rpoB (1447 bp), gyrB (921 bp) and recA (907 bp); 4906 bp and 33 taxa). Redundant sequences were removed from the concatenated data sets. Aligned sequences were converted to FASTA, NEXUS and PHYLIP file formats using ALTER

(http://sing.ei.uvigo.es/ALTER/) (Glez-Peña et al., 2010). The 16S rDNA sequences were aligned using the RDP release 9.55 (Ribosomal Database Project-II) server

(https://rdp.cme.msu.edu) (Cole et al., 2005). Sequences were translated to RNA and corrected manually using the secondary structure of E. coli (J16095) 16S rRNA downloaded from the

Comparative RNA Website and Project (http://www.rna.ccbb.utexas.edu) (Cannone et al., 2002).

Ambiguously aligned regions were excluded from the dataset.

Phylogenetic Analyses

Parsimony analyses were conducted in PAUP v4.0b10 (Swofford, 2002) with all characters equally weighted to find a minimum-length tree (heuristic search, step-wise addition, addition sequence = as is, tree bisection reconstruction (TBR) branch-swapping algorithm). The gaps were treated as missing data and branch support was estimated by bootstrapping using the same tree search parameters (1000 replicates). For the 16S rRNA gene, ambiguous regions

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within the alignment were coded with INAASE (http://lutzonilab.org/news-release-november-

2007/) and included along with the stepmatrices in the parsimony analysis (Lutzoni et al., 2000).

These ambiguous regions were removed from the alignment for Bayesian and maximum likelihood analyses. To test for phylogenetic congruence between gyrB, recA, groEL, and rpoB, the statistical significance of the incongruence length difference (ILD; Farris, et al., 1994, 1995) or partition homogeneity was assessed in PAUP v4.0b10 (Swofford, 2002). For this test 500 replications were performed with heuristic searches (10 random sequence additions and TBR branch swapping). Bayesian and Maximum Likelihood (ML) criteria were used to analyze each individual gene region and the two concatenated data sets. Models of sequence evolution were evaluated using MrModeltest 2.3 (https://github.com/nylander/MrModeltest2) (Nylander, 2004) under the Akaike information criterion (Posada & Buckley, 2004). Bayesian analyses were implemented in MrBayes v3.2.5 and v3.2.6 (http://mrbayes.sourceforge.net) (Huelsenbeck &

Ronquist, 2001; Ronquist & Huelsenbeck, 2003; Ronquist et al., 2012). The reconstructed trees were built with data sets that were partitioned by gene using the SYM + I + G model for the gyrB partition and the GTR + I + G model for all other partitions. Two Markov chain Monte Carlo

(MCMC) analyses were run for each data set with four chains (one cold and three hot). Each analysis ran for 10 million generations (printfreq=1000, samplefreq=100). After the analyses were completed, the application Tracer 1.6 (Rambaut et al., 2014) was used to verify that stationarity had been reached. Trees generated prior to reaching stationarity were discarded as burn-in. ML analyses were conducted with RAxML using both a single threaded version

(v8.2.3) and a parallelized version (v8.2.6) (http://sco.h- its.org/exelixis/web/software/raxml/index.html) (Stamatakis, 2014). Because it is not recommended to estimate the proportion of invariant sites while estimating the Γ parameter

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(RAxML manual), the simpler General Time Reversible model of substitution with a gamma distributed rate variation across sites was applied (GTRGAMMA), and 1000 rapid bootstrap inferences were performed with partitioned data and as well as the combined sequences.

Bayesian and maximum likelihood analyses were performed using the University of Florida

High Performance Computing cluster (http://hpc.ufl.edu/) and the CIPRES Science Gateway

(http://www.phylo.org) (Miller et al., 2010). The program SumTrees v4.0.0 of the DendroPy v4.0.3 package (Sukumaran & Holder, 2010; 2015) was used to build 50% majority rule consensus trees for Bayesian analyses once the burn-in was established. Phylogenetic trees were edited using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree).

Recombination Analyses

Due to the discordance among the phylogenetic trees, investigation of the occurrence of recombination was conducted by the construction of split decomposition graphs and the calculation the pairwise homoplasy index (PHI) (Bruen et al., 2006) using SplitsTree4 v4.14.4

(http://www.splitstree.org) (Huson & Bryant, 2006). GARD (Genetic Algorithm for

Recombination Detection) (Kosakovsky Pond et al., 2006) was implemented using the Adaptive

Evolution Server (http://www.datamonkey.org) (Delport et al., 2010) to search for putative recombinant breakpoints in the alignments. The Tamura-Nei (TrN) model was selected using the local automatic model selection tool along. Gard was executed using the β-Γ site-to-site rate variation and a rate class of four.

Results

Diversity as Assessed by rep-PCR

Reproducibility of the rep-PCR amplification patterns for all strains was verified by repeating all amplifications from two isolates of the same strain a minimum of two times.

Genomic fingerprints were generated from total chromosomal DNA extracted from 59 strains of

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Xenorhabdus and four non-pigmented forms thereof (Table 3-1). For the combined BOX, REP and ERIC amplified sequences, complex patterns were formed for each strain consisting of more than 30 DNA fragments that ranged in size from less than 0.22 kb to more than 8.0 kb.

Pearson’s product-moment correlation (r) values were calculated for the DNA fingerprints revealing distinct clusters for bacteria isolated from specific groups of nematodes (Fig. 3-3).

With the exception of one strain from X. poinarii and one strain from X. bovienii, the bacterial strains within species converged into single groups. With the combined BOX, REP and

ERIC profiles, both X. poinarii NC and X. bovienii OR1 generated unique genomic profiles causing these strains to form clusters with other Xenorhabdus species. X. poinarii NC converged with X. nematophila and X. bovienii OR1 converged with X. indica and X. koppenhoeferi but with r values of less than 0.2 and less than 0.3, respectively. The low r values indicated that these strains are not very similar to the other species within the clusters but rather are unique among the bacterial strains in this study. Analyses of profiles generated from BOX and ERIC rep-PCR separately revealed that X. poinarii NC and X. bovienii OR1 formed clusters with strains of their respective species. This did not occur with the REP generated profiles.

The two representative strains of X. innexi formed a cluster independent from all other

Xenorhabdus strains (r value of < 0.15). Of the strains which had typical and nonpigmented phenotypes (X. hominickii Sm and Smb, X. koppenhoeferi AMK1, AMK1b, AMK2 and AMK2b and X. szentirmaii J1B and J1Bb), X. szentirmaii J1B and J1Bb showed the most difference having some dissimilarities in their patterns within the ERIC generated profiles. X. hominickii

Sm and Smb also displayed differences in the ERIC profile as the profile for Smb was slightly more complex than that of Sm. Any differences among X. koppenhoeferi phenotypes could be attributed to background noise. Unique genomic profiles were generated for the two undescribed

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isolates from Texas (TX10 and TX26), as well as for the undescribed isolate from Vietnam

(VT13). The Texas strains clustered with X. nematophila but are not very similar to X. nematophila (r < 0.25). The strain from Vietnam formed a slightly closer cluster with X. beddingii, but they are also not very similar (r < 0.40).

Phylogenetic Analyses

Partial sequences generated for Xenorhabdus were deposited in GenBank: groES-groEL

(accession numbers FJ533165-FJ533185), gyrB (accession numbers FJ533186-FJ533209), rpoB

(accession numbers FJ533210-FJ533231) and recA (accession numbers FJ536262-FJ536283).

The partition homogeneity test indicated significant discordance among all pairwise comparisons

(P = 0.01). However, when X. poinarii NC was removed from all data sets, groEL became congruent with rpoB (P = 0.48), gyrB became marginally congruent with groEL and rpoB (P =

0.05), and recA remained incongruent with all gene sequences (P = 0.01). Six data sets were generated for this study based on the availability of sequences in GenBank. The first three data sets consisted of 57 taxa with gyrB, recA and gyrB-recA concatenated sequences, and the second three data sets consisted of 29 taxa with groEL, 27 taxa with rpoB and 32 taxa for all four protein sequences combined.

Bayesian analyses were conducted with separate gyrB and recA data sets, and the majority rule consensus trees are presented in Figure 3-4. The gyrB tree was rooted with

Photorhabdus luminescens TT01, Proteus mirabilis HI4320 and Escherichia coli K12, and the recA tree was rooted with P. mirabilis HI4320 and E. coli K12, only. The recA gene for P. luminescens converged with the Xenorhabdus ingroup and was unable to support the tree as part of the outgroup taxa. The two sequences produced dissimilar trees, with a few exceptions. Both reconstructions produced a large monophyly consisting of X. indica, X. cabanillasii, X. budpestensis, X. innexi, X. stockiae and X. bovienii, as well as producing similar monophylies

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among closely related strains. In both tree reconstructions, the position of X. mauleonii is unresolved. X. poinarii NC is positioned within the X. nematophila clade (posterior probability value of 1.0), while all other strains of X. poinarii formed a separate monophyly in both trees.

The recA X. nematophila clade also contains X. doucetiae LWD1 with a posterior probability value of 1.0. In the gyrB generated tree, X. koppenhoeferi is positioned as a sister taxon to the X. nematophila clade; whereas, in the recA generated tree, X. koppenhoeferi is part of a much larger clade. For the gyrB tree the undescribed Xenorhabdus sp. TX26 is a sister taxon to X. koppenhoeferi and part of a larger clade that contains X. szentirmaii, though not well supported; whilst in the recA tree, it is a sister taxon to X. szentirmaii posterior probability value of 1.0. The undescribed species from Vietnam, Xenorhabdus. sp. VN13, formed a clade with X. khoisane and X. miraniensis for the gyrB tree and with X. japonica for the recA gene tree.

However, the clade with X. japonica is unreliable as much of the recA tree is unresolved (low posterior probability values).

The gyrB-recA concatenated Bayesian majority rule and ML trees are presented in Figure

3-5. The trees were rooted with sequences from P. luminescens TT01, E. coli K12 and S. proteamaculans 568. Both analyses produced trees that were similar for most positions.

However; for the concatenated Bayesian analysis, X. bovienii formed a unique clade that is paraphyletic to the rest of Xenorhabdus and no longer part of the large monophyly that was observed with the individual gene and concatenated ML trees. In the Bayesian tree X. doucetiae

LWD1 formed a monophyly with the FRM16 strain of X. doucetiae, but it converged with the X. nematophila clade in the ML tree as it did for the recA Bayesian tree. The clade containing X. beddingii and X. romanii PR06-A remains unresolved as does the position of X. budapestensis among X. indica and X. cabanillasii. Based on the concatenated trees, the undescribed species,

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Xenorhabdus sp. TX-26, is a sister taxon to X. szentirmaii with a posterior probability value of

0.98 but a ML bootstrap value of only 63. The undescribed species, Xenorhabdus sp. VN13, is sister to the clade consisting of X. miraniensis and X. khoisanae with a posterior probability value of 1.0 and a ML bootstrap value of 100.

Bayesian analyses were conducted with the 29 taxa groEL and 27 taxa rpoB data sets, and the majority rule consensus trees are presented in Figure 3-6. The trees were rooted with P. luminescens subsp. laumondii TT01, P. asymbiotica subsp. asymbiotica ATCC 43949, P. mirabilis 528 and E. coli K12. The rpoB analysis also included P. temperata subsp. thracensis in the outgroup. The trees produced from groEL and rpoB are discordant trees except for the positions among closely related taxa and the monophyly consisting of X. indica, X. cabanillasii,

X. budapestensis and X. innexi (posterior probability value of 1.0) and another monophyly consisiting of X. poinarii, X. doucetiae and X. kozodoii. Xenorhabdus sp. TX-26 is positioned within the X. nematophila clade (posterior probability value of 1.0) within the groEL generated tree, but according to the rpoB gene Bayesian analysis, Xenorhabdus sp. TX-26 is positioned into a clade with X. szentirmaii (posterior probability value of 1.0). The undescribed species from Vietnam, Xenorhabdus sp. VN13, formed a clade with X. beddingii in the rpoB tree, but in the groEL generated tree, it is part of a larger clade that includes X. beddingii along with the X. poinarii, X. doucetiae and X. kozodoii monophyletic group. The rpoB sequence for X. poinarii

NC was identical to the sequence of X. poinarii FL224; however, within the groEL generated tree, X. poinarii NC formed a monophyletic group with X. beddingii Q58/1.

Phylogenetic trees for the concatenated gyrB-recA-groEL-rpoB partial gene sequences are presented in Figure 3-7 based on ML (A) and Bayesian (B) methodologies. The trees were rooted with P. luminescens ssp. laumondii TT01, E. coli K12 and Proteus mirabilis HI4320.

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Regardless of the phylogenetic method employed, the trees bifurcated into two groups: one clade contained X. cabanillasii, X. indica, X. budapestensis and X. innexi, which formed a larger monophyly with X. bovienii, and the other contained the rest of Xenorhabdus. Within the later clade, two monophyletic groups were formed: one was between Xenorhabdus sp. TX-26 and X. szentirmaii and the other was between X. doucetiae and X. kozodoii. Some differences in tree topology did occur. For example, there was no congruency between phylogenetic trees for the positions of X. beddingii, X. koppenhoeferi and X. poinarii NC. This was observed with all genes analyzed individually as well. For the Bayesian analysis, Xenorhabdus sp. VN13 formed a monophyletic group with X. beddingii Q58/1 (posterior probability value of 1.0), but in the ML analysis, Xenorhabdus sp. VN13 had an individual lineage (bootstrap value of 79).

For the 16S rRNA sequences, both Xenorhabdus strain TX26 and strain VN13 shared

98.7% sequence identity with the sequence of X. szentirmaii strain SaV. Also, using the RDP seqmatch program Xenorhabdus strain TX26 was given a similarity score of 0.992 to X. mauleonii and strain VN13 given a similarity score of 0.997 to X. szentirmaii strain K77. A phylogenetic tree of Xenorhabdus based on maximum parsimony of partial 16S rDNA sequences is shown in figure 3-8. The tree pictured here is one of four equally parsimonious trees. The differences occurred in the position of Xenorhabdus sp. VN13 and X. poinarii. If X. poinarii was not part of the monophyly that consists of X. kozodoii, X. budpestensis, X. ehlersii, X. innexi, X. indica, X. griffinae and X. ishibashii, it had an independent lineage falling between X. bovienii and the rest of the ingroup taxa. Strain VN13 was either located just outside of the monophyletic group in which it is depicted or it formed a polytomy within the clade. Neither strain TX26 or

VN13 formed a monophyly with the species with which they share the closest sequence identity.

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Bayesian and maximum likelihood analyses were conducted to further assess the relationship of these strains with the other Xenorhabdus species. A maximum likelihood tree is depicted in Figure 3-9. The topology of the tree was very similar to the tree obtained from the

Bayesian analysis. Hence, the posterior probability values are presented where congruent with the ML tree and the value was higher than 0.70. This tree is discordant with the maximum parsimony tree, but like the parsimony tree, the TX26 and VN13 strains do not converge with X. mauleonii or X. szentirmaii.

Recombination

To investigate if the discordance among gene trees was due to recombination, three different methodologies were applied. First, a split decomposition graph using the four concatenated sequences was constructed (Figure 3-10). The graph is nearly tree-like, with the exception of the position of X. poinarii NC and the portion leading to X. bovienii and the monophyly consisting of X. innexi, X. budapestensis, X. cabanillasii and X. indica. There is a thin box leading to X. doucetiae indicating some conflict in the phylogeny. Not surprisingly, there is some reticulation in the position of X. poinarii NC.

In the second method, the pairwise homoplay index was calculated for the concatenated gene set to determine if there was evidence for recombination as well as for individual genes to ascertain if any recombination found could be intragenic (Table 3-4). The PHI test found significant evidence for recombination in the concatenated set (P = 0.0). According to the PHI test there was significant evidence for recombination within the gyrB, groEL and rpoB gene sequences. There was no evidence for recombination within the recA partial sequence.

Accordingly, the gene alignments were analyzed using GARD to search for putative breakpoints, which are presented in Table 3-4. There was no evidence of recombination in gyrB, which contradicts the support of the PHI test. One breakpoint was found in each groEL and

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rpoB sequence alignment. The groEL and rpoB gene sequences were separated into 2 partitions according to their respective breakpoint. These partitions were then analyzed again using the

PHI test to determine if there was a segment free of recombination. Nucleotides 1-684 for the groEL sequence and 671-1447 for the rpoB sequence showed no significant evidence for recombination. The breakpoint found for Photorhabdus in another study using Xenorhabdus within the group found a breakpoint at position 410. This was applied to Xenorhabdus and nucleotides 411-838 gave no evidence of recombination when the PHI test was applied.

A phylogenetic tree depicting the relationships among Xenorhabdus species/strains with the putative recombinant regions removed is shown in Figure 3-11. The topology of the tree does not yield any new information regarding the phylogenetic positions of Xenorhabdus.

The strain VN13 formed a monophyly with X. beddingii, which is supported by the Bayesian tree and is discordant with the ML tree of the concatenated sequences without the regions of potential recombination removed. Also, unlike the previous ML tree, the position of X. poinarii NC is located with X. nematophila and not the X. poinarii clade. The position of X. koppenhoeferi is not within the X. nematophila clade, and the two strains of X. doucetiae diverged into separate clades.

Discussion

This is the first study to demonstrate the utility of rep-PCR using all three BOX, REP and

ERIC primers in the differentiation of Xenorhabdus. Tailliez et al (2006) used rep-PCR for the differentiation of Xenorhabdus strains using ERIC primers that were modified on the basis of the

X. nematophila genome. Generation of the genomic profiles based on ERIC sequences with combined RAPD profiles proved to be a good tool to separate Xenorhabdus strains into species for further analyses (Tailliez et al., 2006). The ERIC primers used here were those of Versalovic et al. (1991) and generated complex profiles that were reproducible. It was observed that with

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the concatenated rep-PCR profiles, Xenorhabdus strains were separated into their respective species, except for one strain of X. poinarii (NC) and the X. bovienii strain isolated from S. oregonense.

The X. poinarii strain NC was shown to be different from other X. poinarii strains with the analyses of all but one of the housekeeping genes. Only the rpoB gene was similar to the other X. poinarii rpoB sequences and identical to X. poinarii strain FL224. It is possible this strain could represent a different species of Xenorhabdus and/or one that has undergone recombination or some other evolutionary event. The 16S rRNA sequence would have to be compared to the sequences of other known Xenorhabdus species to determine if it is indeed unique.

For all other species analyzed, rep-PCR provided an accurate method in determining the bacteria that were most closely related. Two strains, Xenorhabdus sp. TX-26 and Xenorhabdus sp. VN13, generated unique profiles setting these strains apart from the other species included in this study. This was also supported by the phylogenetic analyses. According to the 16S rDNA data, both of these strains are very closely related to X. szentirmaii and Xenorhabdus sp. TX26 is also close to X. mauleonii. The similarity value of 98.7% is currently used as the universal standard for species delineation (Stackebrandt & Ebers, 2006). For other bacteria when using at least 800 bp of rpoB the species cut off is 96-98% similarity (Khamis et al., 2005; Fresno et al.,

2014). Strain TX26 and strain VN13 shared a 96.6% and 93.7% similarity, respectively, with the rpoB sequence of X. szentirmaii. These data indicate that strain TX26 is likely a strain of X. szentirmaii, while strain VN13 could be a new species. Further investigation would need to be conducted to support the data presented here.

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Rep-PCR has been used effectively and efficiently as a method for the differentiation of various species of bacteria (Louws et al., 1994, 1998; Hoffmann & Roggenkamp, 2003; Kaeding et al., 2007). For bacterial species delineation, DNA:DNA homology is the standard and congruence between rep-PCR and DNA reassociation has been demonstrated (Rademaker et al.,

2000). For this study it was a useful tool for separating the strains into various species.

Excluding the aberrant X. poinarii NC strain, X. bovienii Or1 was the sole strain that did not coalesce into a cluster with other strains of its species. However, it did cluster with other X. bovienii using only the profiles derived from ERIC and BOX primers. Still it should be noted that not all strains of X. bovienii clustered together with the individual profiles. Though the resolution of bacteria into their appropriate groups is generally more effective when the genetic fingerprint is derived from all three primers, it may be necessary to look at the profiles from each primer individually when the identity of the bacterium is under question. Rep-PCR should be investigated further to determine if it could be used as an alternative for the more laborious and difficult methods for the characterization of new species in Xenorhabdus.

Though 16S rRNA sequence data is necessary for the description of new species,

Xenorhabdus phylogenetic trees derived from 16S rRNA are incongruent with those derived from housekeeping genes (Lee & Stock, 2010a; Tailliez et al., 2010; Ferreira et al., 2016).

Partial gene sequences for gyrB and recA in combination with other partial gene sequences have been used largely in the delineation of new Xenorhabdus species (Tailliez et al., 2010; 2012;

Kuwata et al., 2013). Either recA (Lee & Stock, 2010a; 2010b) or recA and gyrB (Tailliez et al.,

2012) partial sequences have also been used in combination with other genes to investigate

Xenorhabdus evolutionary relationships. In this study, partial sequences for both of these genes along with two genes that have not been previously used for Xenorhabdus phylogenies, groEL

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and rpoB, were used for evaluating these relationships. Partial and whole gene sequences of groEl have been used successfully in studying Wolbachia (Glowska et al., 2015) and Bartonella

(Chomel et al., 2016), while partial rpoB gene sequences have been used extensively for evaluating evolutionary relationships among other enteric species (Mollet et al., 1997, Adekambi et al., 2008; Behrendt et al., 2015). This is the first study to demonstrate the utility of groEL and rpoB as markers for the construction of Xenorhabdus phylogenies.

All pairwise comparisons showed discordance when the incongruence length difference test was used. Several Xenorhabdus strains were removed from the alignment yielding the same incongruence result until the X. poinarii strain NC was removed from the alignment. Once this strain was removed, all genes but recA were shown to be congruent. Because recA is commonly used as a locus for phylogenetic reconstruction of Xenorhabdus (Tailliez et al., 2010, 2012; Lee

& Stock, 2010a; Thanwisai et al., 2012; Ferreira et al., 2013a; Kuwata et al., 2013; Ferreira et al., 2016), and the partition homogeneity test is not necessarily a strong indicator of congruence

(Yoder et al., 2001), the recA sequences were concatenated with the other gene sequences for phylogenetic analyses.

The phylogenetic trees presented here are similar to other evolutionary trees previously published (Lee & Stock, 2010a; Tailliez et al., 2010; 2012; Ferreira et al., 2013a; 2016). The dissimilarities occurred with the clade containing X. doucetiae and X. romanii (Tailliez et. al.,

2012; Ferreira et al., 2013a; 2016) and the lineage of X. beddingii (Lee & Stock 2010a). Using the combined, gyrB and recA concatenated data set, X. romanii formed a monophyletic group with X. beddingii with strong support (1.0 posterior probability and 93 bootstrap value).

However, using only the 16S rRNA gene sequence, X. romanii formed a monophyletic group with X. doucetiae. The protein coding gene trees presented here could be different because they

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lack the additional sequence data provided in the other studies. The position of X. romanii could not be determined using the four concatenated genes because the sequence data is not available for groEl and rpoB genes.

The concatenated groEL-rpoB-gyrB-recA based tree reconstructions were largely in support of the concatenated gene trees presented by Tailliez et al. (2012) and Ferriera et al

(2016). However, the Bayesian tree presented here was more similar to these maximum likelihood trees, the clearest difference being the position of X. beddingii Q58. Both Tailliez et al. (2012) and Ferreira et al. (2016) used concatenated recA-gyrB-dnaN-gltX-infB partial gene sequences. The genes recA and gyrB were the only genes in common with this study. In the

Bayesian tree presented here, X. beddingii was located outside of the clade, which consisted of X. nematophila, X. koppenhoeferi, X. szentirmaii, X poinarii. X. doucetiae and X. kozodoii (Figure

3-7). In the two aforementioned published trees, X. beddingii was located within this clade.

The phylogeny of Xenorhabdus based on the housekeeping gene sequences does support parts of the reconstructed phylogeny based on 16S rRNA sequences (Tailliez et al., 2010) mostly for very closely related strains, but differences occurred once more taxa were included in the anlayses. Homologous recombination has been detected in the 16S rRNA gene in Xenorhabdus

(Tailliez et al., 2010; Chaston et al., 2011). Therefore, developing an accurate phylogeny based solely on 16S rRNA data can be problematic. Further, the outgroup taxa had to be chosen carefully for the analyses. If Proteus mirabilis, a more closely related genus than the others of the Enteorbacteriaceae (with the exception of the sister genus Photorhabdus), was used as part of the outgroup taxa of the 16S parsimony analysis, many equally parsimonious trees were generated and often the tree could not be rooted with the outgroup. This was also the case when using species of Yersenia. For this study E. coli and Serratia proteamaculans were used without

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the sister taxon Photorhabdus, which resulted in fewer equally parsimonious trees and a better tree score (777 vs. 962). Regardless of the taxa selected for the outgroup, none allowed for good support at deeper nodes. Similar to the 16S rRNA gene, the outgroup taxa had to be modified for the recA derived tree. If Photorhabdus was used in the analysis, it formed a monophyly with X. szentirmaii and much of the relationships among the ingroup taxa were unresolved forming several polytomies. Though there was still not good resolution within the trees using only recA, it was improved without Photorhabdus among the outgroup taxa.

There was some discordance among the sequence data from the protein coding genes, and therefore, the incidence of recombination was investigated. The PHI and GARD analyses did indicate the presence of intragenic recombination within groEL and rpoB gene sequences.

Though the PHI test indicated significant evidence for recombination within the gyrB sequences, the GARD analysis was unable to locate a putative breakpoint. When the breakpoint from the sister taxon Photorhabdus was applied, the PHI test indicated no evidence for recombination in one of the two partitions. The gyrB gene alignment should be analyzed using other tests for recombination. There was no evidence of recombination in the recA gene. All four of the genes are describes as having a low propensity for lateral gene transfer (Lerat et al., 2003), and therefore, the chance of homologous recombination among these genes would be low. However, recombination has been recorded in Xenorhabdus for other genes, and it appears to have occurred within the genes presented in this study as well. Tailliez et al. (2010) found significant recombination within the 16S rRNA gene, and a study conducted by Sergeant et al. (2006) indicated recombination with the serC gene. One might argue that the production of antibiotic compounds and bacteriocins (Furgani et al., 2008; Morales-Soto & Forst, 2011) secreted by

Xenorhabdus inhibit contact with other taxa and therefore lessen the chances of recombination

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occurrences. In most cases this is true, but some insect intestinal bacteria are able to thrive in the presence of Xenorhabdus (Walsh & Webster, 2003). This could make a recombination event feasible.

To determine if the tree topology would change or if the support would increase, the putative recombinant regions were removed from the sequence alignment prior to conducting maximum likelihood and Bayesian analyses. The topology largely remained the same, albeit there were some changes. The evolutionary data here appeared to be more artificial as the two strains of X. doucetiae were part of separate monophyletic groups as observed in the recA- derived tree (Fig. 3-4). It should be noted that the recA gene was the only gene in this study to have no indication of recombination. Perhaps these are not strains of the same species. Also, X. koppenhoeferi was not positioned within the X. nematophila clade. There was strong Bayesian support for this position but not strong bootstrap support. Overall, the support for this tree was only slightly greater than the tree with the putative recombination events. More studies with more sequences would need to be conducted to determine if these are indeed regions of recombination and if removing them from the alignment could lead to a tree that better reflects the evolutionary history of Xenorhabdus.

In summary, rep-PCR proved to be an effective method for separating Xenorhabdus strains and should be considered as a tool to be used alongside phylogenies constructed with multiple genes for the delineation of new species. The utility of groEL and rpoB for the phylogeny of Xenorhabdus was investigated. These partial sequences are useful and should be included in further studies of Xenorhabdus evolution, but the genes should be used in combination with other genes. There was evidence of recombination in all of the protein coding genes but recA, and this should be investigated futher. Finally, the two undescribed strains of

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Xenorhabdus (TX-26 and VN13) isolated from S. texanum and the undescribed Steinernema sp. from Vietnam, respectively should be evaluated further to determine if they represent new species.

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Table 3-1. Xenorhabdus strains used for this study Bacterial strain Nematode host Geographical origin (Reference) source X. beddingi (Akhurst, 1986; Akhurst & Boemare, 1988) Q58/1a Steinernema sp. Australia R. Akhurstb X.bovienii (Akhurst, 1983; Akhurst & Boemare, 1988) XbINT S. intermedium South Carolina, USA K. Nguyenc XbSN S. feltiae SN France K. Nguyenc T27 S. feltiae TT4 ? Certis USA.b T228/1a S. feltiae Tasmania R. Akhurstb T228/1 SFa S. feltiae Tasmania S. Forstb X. budapestensis (Lengyel et al., 2005) Bico1 S. bicornutum 1 Yugoslavia K. Nguyenc Bicoc S. bicornutum 3 Yugoslavia K. Nguyenc X. cabanillasii (Tailliez et al., 2006) M7 S. riobrave 7-12 Mexico K. Nguyenc M8 S. riobrave 8-14 Mexico K. Nguyenc M9 S. riobrave 9-5 Mexico K. Nguyenc Sr4 S. riobrave TX4 Texas, USA K. Nguyenc TX1 S. riobrave 3-8a Texas, USA R. Stuartc TX3 S. riobrave 3-3 Texas, USA R. Stuartc TX4 S. riobrave 3-7 Texas, USA R. Stuartc TX12 S. riobrave 5 Texas, USA R. Stuartc T355 S. riobrave TT Texas, USA Certis USA.b X. doucetiae (Tailliez et al., 2006) LWD1 S. diaprepesi Florida, USA L. Duncanc LWD2 S. diaprepesi Florida, USA L. Duncanc LWD3 S. diaprepesi Florida, USA L. Duncanc X. hominickii (Tailliez et al., 2006) Sm1 S. monticolum Korea H. Kayac X. indica (Somvanshi et al., 2006; Nguyen et al., 2007) SabEG S. abbasi Egypt Egypt K. Nguyenc SabOM S. abbasi Oman Sultanate of Oman K. Nguyenc X. innexi (Lengyel et al., 2005) Ss1 S. scapterisci Uruguay K. Nguyenc T319 S. scapterisci Uruguay Certis USA.b X. koppenhoeferi (Tailliez et al., 2006) AMK1 S. scarabaei AMK001 New Jersey, USA A. Koppenhöferc AMK2 S. scarabaei AMK002 New Jersey, USA A. Koppenhöferc AMK3 S. scarabaei AMK003 New Jersey, USA A. Koppenhöferc EN1 S. scarabai WN3 New Jersey, USA O. Strauchc EN2 S. scarabai WN4 New Jersey, USA O. Strauchc X. kozodoii (Tailliez et al., 2006) Sa1 S. arenarium Russia K. Nguyenc

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Table 3-1. Continued Strain Host Geographical origin Reference (source) X. nematophila (Thomas & Poinar, 1979) XnAll S. carpocapsae All Georgia, USA K. Nguyenc AN6/1a S. carpocapsae DD136 Virginia, USA R. Akhurstb AN6/1 DBa S. carpocapsae DD136 Virginia, USA D. Bowenb XnBrt S. carpocapsae Breton France K. Nguyenc XnC3 S. carpocapsae C3B Arkansas, USA K. Nguyenc XnC4 S. carpocapsae C4A Arkansas, USA K. Nguyenc XnDD136 S. carpocapsae DD136 Virginia, USA K. Nguyenc XnIt S. carpocapsae Italian Italy K. Nguyenc XnKap S. carpocapsae Kapow Poland K. Nguyenc XnMex S. carpocapsae Mexico Mexico K. Nguyenc XnSal S. carpocapsae Sal Indiana, USA K. Nguyenc XnGA S. carpocapsae GA Georgia, USA D. Shapiro-Ilanc XnNJ S. carpocapsae NJ New Jersey, USA D. Shapiro-Ilanc T25 S. carpocapsae All, TT Georgia, USA Certis USA.b X. poinarii (Akhurst, 1983; Akhurst & Boemare, 1988; Cu1 S. cubanum Cuba K. Nguyenc EU6 S. glaseri EU6 Hungary A. Fodorc NC S. glaseri NC North Carolina, USA K. Nguyenc FL224 S. glaseri 224 Florida, USA This studyc FL446 S. glaseri 446 Florida, USA This studyc X. szentirmaii (Lengyel et al., 2005; Nguyen et al., 2006) 17C S. rarum LA Louisiana, USA K. Nguyenc J1B S. rarum MS Mississippi, USA K. Nguyenc Xenorhabdus sp. Or1 S. oregonense Oregon, USA K. Nguyenc Xenorhabdus sp. TX10 S. texanum 10 Texas, USA K. Nguyenc TX26 S. texanum 26 Texas, USA K. Nguyenc Xenorhabdus sp. VN13 Steinernema sp 13 Vietnam K. Nguyenc Xenorhabdus sp. Jpn Steinernema sp. ? IAM Culture Collectionb aType strain bProvided bacteria cProvided host nematode

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Table 3-2. Sequences of primers used to amplify and sequence gene regions Gene Primer Typea Sequence (5'-3') gyrB gyr-320b Amp/Seq TAARTTYGAYGAYAACTCYTAYAAAGT rgyr-1260b Amp/Seq CMCCYTCCACCARGTAMAGTTC recA recAF Amp/Seq CGAAGGAGTAAACATGGCTAACGA recAR Amp/Seq CTGGATGCTCTTTCAGGTAAATCG groEL groExFc Amp/Seq ATGAAWATTCGTCCRTTRCAYGAYCG groExR1c Amp/Seq TTACATCATKCCRCCATRCCACCCA groR1 Seq GCRCCCATRTTYTCGAAYTTGTC groELIF1 Seq GTAGCACGTGAAATCGAATTGG groELIF2 Seq GCATACCTSTCTCCTTAYTTCATC groELIF3 Seq GCAGCAACTGAACAGTACGGC rpoB rpoF Amp/Seq TGAGCCARTCTGGYCAYAARTCTAT rpoR Amp/Seq TCACGRGAYACRCAMGCCAGTTC d CM7 Seq AACCAGTTCCGCGTTGGCCTGG rpoIF Seq GTATGTCCWATCGAAACSCCTG rpoIR Seq CTTCAGGSGTTTCRATWGGAC Degenerate nucleotides: W = A or T; R = G or A; Y = C or T; K = G or T; S = G or C aAmp, amplification; Seq, sequencing bDauga, 2002 cFares et al., 2002 dMollet et al., 1997

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Table 3-3. GenBank accession numbers for additional gene sequences used in the present study Bacterial strain gyrB recA 16S rRNA Complete genomea X. beddingii Q58 EU934516 FJ823415 AY278675 - X. bovienii CS03 EU934529 FJ823429 - - X. bovienii SS-2004 - - - FN667741 X. bovienii TB20 EU934527 FJ823430 - - X. bovienii TB30 - - DQ208306 - X. bovienii USNY95 EU934528 FJ823427 DQ205453 - X. bovienii - - AY660027 - X. budapestensis CN03 EU934536 FJ823419 DQ211714 - X. budapestensis DSM16342 EU934535 FJ823418 AJ810293 - X. cabanillasii USTX62 EU934537 FJ823422 AY521244 - X. doucetiae FRM16 - - DQ211709 FO704550 X. doucetiae FRG30 - - DQ211702 - X. ehlersii DSM16337 EU934524 FJ823398 AJ810294 - X. ehlersii KR02 JQ687365 JQ687364 DQ208308 - X. griffiniae ID10 EU934525 FJ823399 DQ211710 - X. hominickii KE01 EU934517 FJ823410 - - X. hominickii KmYb11 - - AB507815 - X. hominickii KR05 - - DQ205449 - X. indica DSM17382 EU934538 FJ823421 AM040494 - X. indica OM01 - - DQ211718 - X. innexi DSM16336 EU934540 FJ823423 AJ810292 - X. innexi UY61 - - AY521243 - X. ishibashii AB630948 AB630947 AB243427 - X. ishibashii GDh7 JQ348907 JQ348906 GQ149086 - X. japonica DSM16522 EU934513 FJ823400 DQ202310 - X. japonica - - AB243426 - X. khoisanae SF80 JX623970 JX623967 JX623966 - X. khoisanae SF106-C JX623976 JX623973 JX623972 - X. khoisanae SF362 JX623982 JX623979 - - X. koppenhoeferi UDNJ01 - - DQ205450 -

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Table 3-3. Continued Bacterial strain gyrB recA 16S rRNA Complete genomea X. kozodoii ES01 EU934521 FJ823405 - - X. kozodoii FR48 - - EU190977 - X. kozodoii IT10 EU934523 FJ823406 - - X. kozodoii SaV EU934522 FJ823404 DQ211716 - X. magdalenensis IMI 397775 JF798402 JF798401 HQ877464 - X. mauleonii VC01 EU934533 FJ823417 DQ211715 - X. miraniensis Q1 EU934520 FJ823414 DQ211713 - X. nematophila ATCC 19061 - - - FN667742 X. nematophila AN6/1 - - - LN681227 X. poinarii AZ26 EU934544 FJ823408 DQ211703 - X. poinarii G6 - - FO704551 X. poinarii CU01 - - DQ211706 - X. romanii PR06-A EU934515 FJ823403 DQ211717 - X. stockiae TH01 EU934542 FJ823425 - - X. stockiae SRK7 - - FJ006728 - X. stockiae HN xs01 - - HQ840745 - X. szentirmaii DSM 16338 CBXF010000099b CBXF010000046c AJ810295 - X. szentirmaii K77 - - DQ211712 - X. vietnamensis VN01 EU934514 FJ823401 DQ205447 - X. sp. TZ01 JQ687370 JQ687369 - - Escherichia coli strain CFT073 - - - AE014075 Escherichia coli strain K12 - - - U00096 substrain MG1655 Photorhabdus luminescens - - - BX470251 subsp. laumondii TT01 Proteus mirabilis strain HI4320 - - - AM942759 Serratia proteamaculans 568 - - - CP000826 aWhere accession number for complete genome provided for complete genome sequences gyrB and recA as well as, groEL, and rpoB were used. bContig 4, whole genome shotgun sequence. cContig 14, whole genome shotgun sequence.

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Table 3-4. Pairwise homoplasy index test (PHI) and Genetic algorithm recombination detection (GARD) to screen for recombination GARD Gene No. of No. of PHI Putative Model averaged Gene fragment L/RHS P- PHI P-value of nucleotides Taxa P-value breakpoint support at breakpoint valuea gene fragment gyrB 838 55 1.7E-5 No evidence - 1-410b - 0.001 411-838b - 0.615 recA 646 46 0.497 No evidence ------groEL 1606 29 0.038 684 0.5024 1-684 0.0002 0.692 685-1606 0.0060 0.002 rpoB 1447 27 0.012 670 0.2405 1-670 0.0038 0.046 671-1447 0.0004 0.806 aL-adjusted KH P-value indicating the topology inferred from the data to the left of the breakpoint is significantly different to that inferred by the data to the right. R-adjusted KH P-value indicating the topology inferred from the data to the right of the breakpoint is significantly different to that inferred by the data to the left. bNo breakpoints were found on this alignment using GARD. This breakpoint was determined for the sister taxon Photorhabdus in another study while Xenorhabdus was included in the analysis.

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gyrB rpoB 0.09 94.82

groEL 78.74 X. nematophila ATCC 19061 recA 4432590 bp 25.40

Figure 3-1. Circular genomic map of X. nematophila with positions of genes used in this study.

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A 1 294 1 1647 groES groEL groExF1 groELIF1 groELIF2 groELIF3 1 166 592 935 190 1647 groR1 groExR1 B 1 1068 recA recF -13

943 recR C 1 2415 gyrB gyr-320 306

1276 rgyr-1260 D 1 4029 rpoB rpoF CM7 rpoIF 974 1384 1672

1697 2525 rpoIR rpoR E 1 1545 1 2909 16S rRNA ITS 23S rRNA FGPS6-63 HK1 6 1029

1536 132 HK2 FGPL132-38 Figure 3-2. Regions of genes selected for this study with locations of PCR and sequencing primers.

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r x 100 20 40 60 80 100 BOX REP ERIC

Ss1 X. innexi T319 TX10 Xenorhabdus sp. TX26 XnC4 XnSal XnC3 XnDD136 XnGA XnNJ XnBrt ATCC 19061 X. nematophila AN6/1 DB AN6/1 XnIt XnMex XnKap XnAll T25 Jpn Xenorhabdus sp. NC X. poinarii Sm X. hominickii Smb Sa1 X. kozodoii M7 M8 TX3 TX12 TX4 Sr4 X. cabanillasii T355 M8 TX1 T382 Bico1 X. budapestensis Bico3 LWD2 LWD1 X. doucetiae LWD3 XbSN T27 T228/1 X. bovienii T228/1 SF XbINT J1B J1Bb X. szentirmaii 17C Q58/1 X. beddingii VN13 Xenorhabdus sp. FL224 FL446 X. poinarii EU6 Cu1 AMK2 AMK2b AMK1 AMK1b X. koppenhoeferi AMK3 EN2 EN1 SabOM X. indica SabEG OR1 Xenorhabdus sp. Figure 3-3. Cluster analysis of banding patterns of Xenorhabdus strains. Genomic fingerprints were generated by rep-PCR using primers corresponding to BOX1A, REP and ERIC sequences. The dendrogram was constructed with UPGMA with similarity levels expressed as the percentage values of the Pearson product-moment correlation coefficient (r).

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A B

Figure 3-4. Phylogenetic relationships between species/strains of Xenorhabdus based on Bayesian analysis of partial gyrB and recA sequences. Majority rule consensus trees constructed using MrBayes, 10,000,000 generations, 10% burn-in. A) gyrB under the SYM+I+G model. B) recA under the GTR+I+G model. Trees rooted with E. coli K12 and P. mirabilis HI4320. Root for gyrB included P. luminescens laumondii TT01 (root not shown). Posterior probabilities (>0.70) presented as percentages.

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A 8 7 X. ehlersii DSM 16337 B 8 4 X. ehlersii KR02 8 6 X. griffinae ID10 9 9 Xenorhabdus sp. TZ01 100 X. ishibashii GDh7 9 9 X. ishibashii X. kozodoii IT10 X. kozodoii ES01 100 X. kozodoii SabOM X. kozodoii SaV X. doucetiae FRM16 X. magdalensis IMI 397775 100 X. beddingii Q58/1 9 3 X. beddingii Q58 GB X. romanii PR06-A 100 X. japonica DSM16522 X. vietnamensis VN01 9 2 X. poinarii GK 9 1 X. poinarii AZ26 8 7 X. poinarii FL224 100 X. poinarii G6 X. poinarii Cu1 8 0 X. nematophila DD136 X. nematophila ATTC 19061 7 9 X. poinarii NC 9 6 X. nematophila XJ 7 1 X. nematophila ScC4 X. doucetiae LWD1 X. koppenhoeferi AMK1 9 9 X. szentirmaii DSM16338 100 X. szentirmaii 17C X. szentirmaii J1B Xenorhabdus sp. TX26 X. mauleonii VC01 100 X. hominickii Sm1 X. hominickii KE01 9 9 X. khoisanae SF106-C 100 X. khoisanae SF80 100 X. khoisanae SF362 100 X. miraniensis Q1 Xenorhabdus sp. VN13 100 X. indica SabOM 100 X. indica SabEG 9 8 X. indica DSM17382 100 X. cabanillasii Sr4 100 X. cabanillasii USTX62 X. budapestensis Bico1 9 9 X. budapestensis DSM16342 100 X. budapestensis CN03 100 S. innexi DSM16336 100 S. innexi Ss1 X. stockiae TH01 X. bovienii Or1 X. bovienii XbINT X. bovienii USNY95 9 9 X. bovienii XbSN 100 X. bovienii CS03 9 2 X. bovienii T228/1 X. bovienii TB20 Figure 3-5. Maximum likelihood tree and Bayesian majority rule consensus tree of the combined gyrB and recA sequences. A) ML analysis conducted in RaxML using GTRGAMMA. B) Bayesian analysis conducted with MrBayes. Analysis run for 10,000,000 generations, sample frequency = 100. The gyrb partition was run under the SYM+I+G model and the recA partition run under GTR+I+G model. The first 10% of trees were discarded as burn-in. Trees rooted with E. coli K12, P. mirabilis HI4320 and P. luminescens laumondii TT01 (root not shown).

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A B

Figure 3-6. Bayesian analysis of the groEL and rpoB partial sequences, majority rule consensus trees. A) groEL. B) rpoB. The analyses were run for 10,000,000 generations, sample frequency = 100 under the GTR+I+G model. The first 10% of trees were discarded as burn-in. Posterior probabilities are presented as percentages. Trees rooted with E. coli K12, P. mirabilis HI4320 and P. luminescens laumondii TT01 (root not shown).

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Figure 3-7. Phylogenetic analyses of the combined gyrB, recA, groEL and rpoB sequences. A) Phylogram of the best ML tree (−ln = 4350.2744) generated using RaxML under the model GTRGAMMA for each partition. B) Bayesian tree was run for 10,000,000 generations, sample frequency = 100. The gyrb partition was run under the SYM+I+G model and the other the partitions run under GTR+I+G model. The first 10% of trees were discarded as burn-in. ML bootstrap support values and posterior probabilities as percentages above branches of respective trees.

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Figure 3-8. Maximum parsimony analysis of 16S rDNA partial sequences. One of four equally parsimonious trees. The undescribed species are highlighted in light blue. Tree rooted with E. coli strain K12 and S. proteamaculans strain 568 (not shown).

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Figure 3-9. Phylogenetic relationships between species/strains of Xenorhabdus based on maximum likelihood analysis of partial 16S rRNA gene sequences constructed in RAxML. Bootstrap support values greater than 70 are presented above branches. Numbers below branches represent posterior probability as percentages. RAxML conducted under the GTRGAMMA model and the Bayesian analysis under the GTR+I+G model in MrBayes (10,000,000 generations, 10% burn-in). The undescribed species are highlighted in blue. Tree rooted with E. coli strain K12 and S. proteamaculans strain 568.

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Figure 3-10. Split decomposition graph of concatenated housekeeping genes for Xenorhabdus strains. The graph was constructed using concatenated gyrB-recA-rpoB-groEL DNA sequences using the uncorrected P characters, fit score is 73.93.

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Figure 3-11. Maximum likelihood tee of concatenated gyrB-recA-rpoB-groEL with putative recombination sites recombination removed. ML tree constructed using RAxML under the GTRGAMMA model of nucleotide substitution for each partition. Bootstrap values located above branches. Posterior probability support presented as percentages located under branches. Bayesian analysis conducted in MrBayes using the GTR + I + Γ model for each partition. Analysis run for 10,000,000 generations, sample frequency = 100, and burn-in set for 25%. Tree rooted with E. coli K12 and P. mirabilis HI4320.

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CHAPTER 4 A STUDY OF THE 16 S rRNA IN Xenorhabdus spp.

Introduction

The 16S (small subunit) rRNA gene is often used for phylogenetic studies of prokaryotes and is required for the delineation of new species. This gene has its limitations for phylogenetic inference. Though it is rare, ribosomal genes can undergo horizontal gene transfer across varied taxonomic groups, or these genes can undergo recombination events, which may lead to phylogenetic data that is misleading (Yap et al., 1999). Accordingly, resolving a bacterial evolutionary history based solely on 16S rRNA gene sequences is not reliable (Lerat et al.,

2003). It has been suggested that in Photorhabdus, the sister taxon to Xenorhabdus, the 16S rRNA genes may be present as a mixture of paralogous rather than orthologous genes (Peat et al., 2010). As in Photorhabdus, multiple copies of this gene exist within the Xenorhabdus chromosome. Phylogenies of Xenorhabdus, which are constructed using the 16S rRNA gene, often produce trees that are discordant with those created by using housekeeping genes (Tailliez,

2010; 2012). Though 16S rRNA is sufficient for evaluating these bacteria at the genus level, it may not be adequate for determining the positions of bacterial strains at species and subspecies levels. The current standard for species determination is 98.7% similarity for the 16S rRNA gene, but there are distinct species, which differ phenotypically, that share a higher degree of similarity (Nguyen et al., 2016).

The objectives of this study were to look for disparity among copies of the 16S rRNA gene within individual isolates and to determine the significance of the secondary structure for species determination. Multiple copies of the 16S rRNA gene were evaluated for sequence similarity within the isolate itself and among several Xenorhabdus strains, and these sequences were aligned according to their secondary structure. Areas among the loop regions would be

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evaluated to determine if the more ambiguous regions tend to be more species specific. With these more specific alignments, perhaps a more robust phylogeny for Xenorhabdus can be determined.

Materials and Methods

Isolation of Bacterial Strains

Symbiotic bacteria were liberated from entomopathogenic nematode hosts maintained in the Nematode Evolution Laboratory at the University of Florida, Gainesville and from those supplied by Dr. Larry Duncan (University of Florida) and Dr. Albrecht Koppenhöfer (Rutgers

University). To isolate the bacterial symbionts, infective stage juveniles (IJs)were surface sterilized by incubating for 30 min in a solution of 0.125% (w/v) methylbenzathonium chloride with gentle rocking, followed by a 15 min incubation in 3% (v/v) hydrogen peroxide. The IJs were rinsed three times with sterile tap water and macerated to release their symbiotic bacteria.

Xenorhabdus strains were cultured on Tergitol-7 Agar (BBL, Becton Dickinson and Company,

Franklin Lakes, NJ) supplemented with 0.004% triphenyltetrazolium chloride and identified by their morphological growth characteristics. Additional bacteria were provided by Dr. Ray

Akhurst (CSIRO Entomology, Canberra, Australia) and Dr. Steve Forst (University of

Wisconsin, Milwaukee). Details of the origin and nematode host affiliation of the strains used in this study are presented in Table 4-1.

Preparation of Genomic DNA

Cultures were grown overnight in 5 ml of Luria-Bertani broth at 27°C with shaking at

150 rpm. The cells were collected by centrifugation of 1 ml of the broth culture at 8,000 rpm for

3 min. The resultant pellet was washed with 500 µl of sterile tap water, and the cells were again collected by centrifugation. Total genomic DNA was prepared by a modified method of Ausubel

(1992). Bacterial cells were incubated in 600 µl cell lysis buffer (10 mM Tris-HCl, pH 8.0; 1

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mM EDTA; 0.5% (w/v) sodium dodecyl sulfate; 30 µg proteinase K) for 1 h at 37°C. The DNA was purified from the cell lysates by incubation with 150 µl of 5 M sodium chloride and 80 µl

8.0; 1 mM EDTA). The DNA was quantified spectrophotometrically.

The 16S rDNA Sequence

The primers that were used for amplification and sequencing are presented in Table 4-2.

The 16S small subunit, the internal transcribed spacer region and part of the 23S large subunit were amplified using the primers of Ponsonnet & Nesme (1994). Each reaction mixture had a total volume of 50 l containing 1 × PCR buffer, 2.5 mM MgCl2, 0.2 µM each deoxynucleoside triphosphate, 1 µM each forward and reverse primer, 1 U Taq polymerase (Promega

Corporation, Madison, WI) and 1 l DNA template. The thermal cycling program consisted of an initial denaturation step for 4 min at 95C, 30 cycles of denaturation at 94C for 30 s, annealing at 52C for 1 min and extension at 72C for 2.5 min and a final extension step at 72C for 10 min. Multiple rRNA cistrons were amplified, which in most cases could be distinguished by the size of the amplified product. Products with a larger molecular weight generally had a larger internal transcribed spacer region. The amplified products were extracted from the agarose gel and then cloned and sequenced.

The rDNA PCR products were ligated into a T-overhang vector using the pGEM®-T

Vector System (Promega Corporation). The plasmid was either transformed into MAX

Efficiency® DH5α™ Competent Cells (Invitrogen, Carlsbad, CA), or the TOPO TA Cloning®

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kit (Invitrogen) was used according to the manufacturer's instruction. Colony-PCR was used to verify the presence of cloned inserts. Colonies that contained inserts were grown overnight in

LB broth containing ampicillin and purified using the QIAprep Spin Miniprep Kit (Qiagen, Inc.,

Valencia, CA).

The gene products were then sequenced using the ABI BigDye® Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) using an amended protocol. Each sequencing reaction contained 2.0 l ICBR mix (proprietary, University of Florida), 2.0 l

BigDye® ready reaction mix, 3.2 pM sequencing primer, 350 ng plasmid template and sterile distilled water to a final reaction volume of 10 l. The thermal profile for sequencing was used as follows: denaturation at 96C for 30 s, primer annealing at 50C for 15 s and product extension at 40C for 4 min for a total of 25 cycles and then held at 4 C. The products were purified by ethanol precipitation. Briefly, each resulting product was placed into a 1.5 ml microcentrifuge tube containing 30.0 l of cold 95% ethanol with 1.0 l 3 M sodium acetate, pH

5.2, incubated on ice for 10 min and centrifuged at 12,000 x g for 15 min. The supernatant was removed, and the resulting pellet was rinsed with 250 l of 70% ethanol and dried using a rotary evaporator. Purified sequencing reaction products were recorded at the University of Florida

ICBR DNA Sequencing Facility (Gainesville, FL) with an ABI 3130 automated DNA sequencer

(Applied Biosystems).

Alignments

The 16S rDNA sequences were aligned with other Xenorhabdus sequences acquired from

GenBank (Table 4-3) using the RDP release 9.55 (Ribosomal Database Project-II) server

(https://rdp.cme.msu.edu) (Cole et al. 2005). The 16S rDNA sequences were translated to RNA and optimized manually using MacClade 4.06 (Maddison & Maddison, 2002) according to the

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secondary structure of E. coli (J16095)16S rRNA downloaded from the Comparative RNA

Website and Project (http://www.rna.ccbb.utexas.edu) (Cannone et al., 2002). Regions of RNA that were ambiguously aligned were recoded as single multistate characters using INAASE 3.0

(Lutzoni et al., 2000) for parsimony-based analyses. The regions were excluded from the dataset for all other analyses.

Equally weighted parsimony analysis was conducted using PAUP v4.0b10 (Swofford,

2002) to find a minimum-length tree (heuristic search, step-wise addition, addition sequence = as is, tree bisection reconstruction (TBR) branch-swapping algorithm). Gaps were treated as missing data. Branch support was estimated by bootstrapping using the same tree search parameters (500 replicates). Prior to Bayesian analysis, the best fit model of nucleotide substitution was evaluated using JModelTest 2.1.7 (Guindon & Gascuel, 2003; Darriba et al.,

2012) with Akaike weights (Posada & Buckley, 2004). Because not all models obtained from

JModelTest can be implemented in MrBayes, three substitution schemes were used to test for the best of 24 models. Bayesian analyses were executed in MrBayes 3.2.6

(http://mrbayes.sourceforge.net) (Huelsenbeck & Ronquist, 2001, Ronquist & Huelsenbeck,

2003). Two Markov chain Monte Carlo (MCMC) analyses were run with four chains (one cold and three hot) for 20 million generations (printfreq=1000). After the analyses were completed, the application Tracer 1.6 (Rambaut et al., 2014) was used to verify that stationarity had been reached. Trees generated prior to reaching stationarity were discarded as burn-in. Both PAUP* and MrBayes analyses were conducted using the CIPRES Science Gateway (Miller et al., 2010).

Pairwise homoplasy index (PHI) values (Bruen et al., 2006) for the Xenorhabdus 16S rDNA alignments and a split decomposition graph was constructed using SplitsTree4 v4.14.4

(http://www.splitstree.org) (Huson & Bryant, 2006).

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Results

Sequences of the nearly complete 16S rRNA small subunit, the internal transcribed spacer region and part of the 23S rRNA large subunit generated for Xenorhabdus were deposited in GenBank (accession numbers FJ515800-FJ5515824). Two additional sequences are presented in Appendix A. Amplifying part of the 23S rDNA allowed for varying sizes of the internal transcribed spacer (ITS) region so that different RNA cistrons could be selected. Most of the sequences amplified contained only tRNA-glutmatic acid (tRNA-Glu) within the ITS. However, for X. budapestensis 17C, X. kozodoii Sa1, and X. bovienii Or1 ITS regions containing tRNA-

Glu as well as those containing tRNA-isoleucine (tRNA-Ile) and tRNA-alanine (tRNA-Ala) were obtained. For X. koppenhoeferi two different ITS products were generated and contained either tRNA-Ile and tRNA-Ala or tRNA-Ile alone.

The different copies of the16S rRNA within the same genome only differed by a single nucleotide for X. budapestensis, X. indica and Xenorhabdus sp. TX26. The sequences differed by two nucleotides for X. hominickii Sm1, and three nucleotides for X. szentirmaii 17C and X. poinarii FL224. Therefore, the similarity of the 16S rRNA isolated within each bacterium had a similarity of 99.8% or greater. The similarity of the 16S rRNA sequences isolated from X. bovienii Or1 ranged from 98.9% to 99.7%. The similarity of the two copies from X. kozodoii was 99.21%, from X. koppenhoeferi was 99.67%, and from X. doucetiae 98.68%. The 16S rDNA portions of the sequences were compared to other 16S rDNA sequences obtained from

GenBank (Table 4-3). The similarity of two undescribed species (VN13 and TX26) is presented in Table 4-4. Both of the sequences are quite similar to X. szentirmaii within the range of species designation which 98.7% or higher. In the case of X. doucetiae, one copy (D-2B) was close to X. doucetiae FRG30 (99.12%) while the other copy (D-2P) was more similar to X. romanii PR06-A

(99.39%).

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The sequences were highly conserved, but there were three regions of sequence ambiguity where the 16S rRNA sequences could not be aligned with confidence and are presented in Figure 4-1. These nucleotide positions were coded by INAASE prior to parsimony analysis, resulting in step matrices ranging from four to seven steps. The parsimony tree is presented in Figure 4-2. The ambiguous regions were discarded from the alignment prior to nucleotide substitution model selection and Bayesian analysis. The Bayesian tree is presented in

Figure 4-3.

Regardless of phylogenetic method employed, monophyletic groups were formed between X. budapestensis, X. ehlersii, X. kozodoii, X. innexi and X. stockiae; X. doucetiae, S. romanii, X. nematophila, X. magdalensis, X. beddingii and X. cabanillasii; X. ishibashii and X. griffinae; X. hominickii and X. khoisanae; and X. japonica and X. vietnamensis. Xenorhabdus bovienii has the most external position of the ingroup taxa in both trees. Though the trees are similar in regards to closely related taxa, there are differences at the deeper nodes and the parsimony tree is not well supported at these nodes. For the Bayesian derived tree, X. poinarii is part of a monophyletic group with X. ishibashii and X. griffinae (posterior probability value 1.0), and X. indica forms a monophyly with this group (posterior probability value 0.99). For the parsimony tree, though they are part of a larger monophyly consisting of X. ishibashii and X. griffinae and other species, X. indica and X. poinarii have independent lineages (bootstrap support value low). The X. doucetiae LWD1 D-2P 16S rDNA sequence formed a monophyly with X. romanii and the LWD1 D-2B sequence was independent of both X. doucetiae and X. romanii.

The pairwise homoplasy index was calculated for 16S rDNA data set to determine if there was evidence for recombination. The PHI test found significant evidence for

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recombination in (P < 0.0001). The entire data set was analyzed as well as only the 16S rDNA that was sequenced for this study. This data set also showed significant evidence for recombination (P = 0.00737). Once the two rRNA sequences from the X. doucetiae, D-2B and

D-2P, were removed from the data set, there was no evidence of recombination (P = 0.2121).

The split decomposition graph is illustrated in Figure 4-4. The graph is largely tree-like, with the exception of X. doucetiae. The parallel edges indicate discordance with the phylogenetic signal.

Discussion

The 16S rRNA sequences were highly similar with some areas of variability; specifically, five small regions with higher levels of variability were identified. These regions corresponded with the variable regions one, two, three, seven and nine within the secondary 16S rRNA structure as described by Yarza et al. (2014). Three of these regions were ambiguously aligned due to the presence of nucleotide insertions when compared to the 16S rRNA of Escherchia coli.

Unlike their sister taxon, Photorhabdus, which has a characteristic insertion of nucleotides in the loop of H199 of the variable region two, there were no genus specific insertions.

The first area of significant sequence variation occurred in variable region V1, the loop of helix H61 (Cannone et al., 2002). This is considered the most variable region of the16S rRNA structure (Yarza et al., 2014). With only a few exceptions the nucleotide differences appeared to be species specific among the representatives in this data set. The exceptions included a single nucleotide difference in one strain from each X. poinarii and X. bovienii. Also, one 16S rRNA copy from X. doucetiae LWD1 D-2P shared sequence homology with that of X. romanii, while

LWD1 D-2P was the same as the other X. doucetiae strains. This gene copy D-2P was highly homologous with that of X. romanii.

Within the variable region V2 of Yarza et al., (2014) the nucleotide region between the stems and loops of H144 and H184 (Cannone et al., 2002) had two nucleotide variations, one

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that occured at nucleotide position 181 and the other at nucleotide position 183. The variation at position 181, which was a guanine instead of adenine, although present within most representatives of a particular species, was not necessarily species specific. The uracil at position 183 instead of cytosine occurred within all representative strains of X. poinarii and X. doucetiae, suggesting that this might be conserved within particular species of Xenorhabdus.

The nucleotide variant was also present in X. beddingii, X. griffinae and X. romanii, but there was only was representative strain for each of these species. With the exception of X. beddingii, the nucletotides within the stem region were conserved among the Xenorhabus in this dataset.

The loop was highly variable. One other variant that could be conserved among different

Xenorhabdus species was the presence of guanine at nucleotide position 188. This variant occurred in all representative strains of X. japonica, X. indica and X. doucetiae. It also occurred in X. vietnamensis, X. magdalensis and X. romanii, but these species each had only one representative strain.

The third region with exceptional variability occurred in the distal part of the stem and in the loop of H440 (Cannone et al., 2002) of variable region V3 (Yarza et al., 2014). Unlike the other variable regions, there were no insertions, and though the stem had different nucleotides, the secondary structure was conserved. Therefore, this portion of the sequence was easily aligned. While the sequence was homologous for many of the strains within a species, these differences were not always species specific. In fact, several of the strains with multiple 16S rRNA gene copies had variation of the sequence data between gene copies, including X. poinarii

G6, X. nematophila ATCC19061, X. szentirmaii 17C and Xenorhabdus sp. VN13.

In the loop of H1118 (Cannone et al., 2002) in variable region V7 (Yarza et al., 2014), the variation remained within species of this dataset with the exception of the 16S rRNA gene

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copy O-2B of X. bovienii Or1 and the 2 copies from Xenorhabdus sp. VN13. One other area of variability, which caused the sequences to be ambiguously aligned, was in the loop of

H1399(Cannone et al., 2002) in variable region V9 (Yarza et al., 2014). Most strains shared the same sequence as E. coli J10695 (UUCG). Of the 71 sequences in this dataset, only 13 diverged from the E. coli sequence. The differences that did occur in the loop were highly conserved among species within this dataset. Regrettably, for X. cabanillasii, X. romanii, X. mauleionii, X. magdalenensis and X. beddingii there was only one copy of the 16S rRNA gene in the data set.

It is impossible to determine how conserved these regions are within these species. This should be reanalyzed with additional sequence data from GenBank.

The gold standard for species delineation is 70% DNA-DNA hybridization, but 16S rRNA similarity of 98.7% or higher is an indication of the same species (Stackebrandt & Ebers,

2006; Rossi-Tamisier et al., 2015). However, this is an artificial cut-off and several other species do not keep to this standard. Some examples thereof that share a 16S rRNA gene sequence similarity greater than 99% are different species of Edwardsiella (Janda & Abbott,

2007) and different Bacillus species (Fox et al., 1992).

Two undescribed species presented here, Xenorhabdus sp. TX26 and Xenorhabdus sp.

VN13, both share 16S rRNA gene sequence similarity with X. szentirmaii within the species cut off range. However, both phylogenetic trees indicated that neither Xenorhabdus sp. TX26 nor

Xenorhabdus VN13 were strains of X. szentirmaii, but rather their placement in the tree could indicate that they are novel species. Additionally, they were not highly similar to each other

(98.02%). This study also showed that one isolate (X. doucetiae) contains different copies of the

16S rRNA gene that are similar to more than one species. Depending upon the gene that is picked for species delineation, the results could lead to misidentification. The taxon’s position

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within an evolutionary tree may be misleading depending upon which copy of the gene is used to build the phylogeny.

Tailliez et al. (2010) showed that recombination was occurring in the 3’ end of 16S rRNA gene among X. bovienii, X. hominickii, X. innexi, X. nematophila and X. stockiae. The potential for recombination was also presented here. Though more analyses would need to take place to confirm, the results of the PHI test suggest that X. doucetiate LWD1 is somehow involved in recombination. The fact that both copies of the gene were less than 98.7% similar to each other suggests that one gene copy may have been acquired horizontally. This has also been suggested for Photorhabdus (Peat et al., 2010). It should be noted that the sequences were evaluated at the Ribosomal Database Project, and no chimaeras were found. The symbiotic relationship of the bacterium with the host nematode is very specific, and the bacteria live out their life within the nematode or within the insect. One might expect that the brief time in the insect with other bacteria either by co-infection with another nematode or exposure to the insect gut bacteria could potentially subject the bacteria to other species where genetic information could be shared. Perhaps highly similar Xenorhabdus strains are able to share a nematode host, where they can transfer DNA and eventually outcompete the other strain.

One objective was to look for disparity among copies of the 16S rRNA gene within individual isolates. For most of the isolates, the sequences were highly conserved. Three of the copies within the Or1 strain of X. bovienii isolated from the nematode S. oregonense were highly similar to each other and highly similar to the SS-2004 strain isolated from S. jollieti. One 16S rRNA gene copy, O-2B was the least similar and more closely related to the CS03 strain isolated from the nematode S. weiseri. The only truly significant disparity was between the 16S rRNA copies from X. doucetiae, and though highly similar the two gene copies from Xenorhabdus sp.

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VN13 diverged within both the parsimony and Bayesian trees. The gene sequences among

Xenorhabdus are very conserved making it difficult to tease out deeper nodes. The few ambiguous regions were recoded with hopes to gain more discriminatory power, but it didn’t seem to improve the resolution of the phylogenetic trees. Perhaps the sequence can be narrowed down to contain variable regions with less conserved regions.

The16S rRNA gene has been shown to be inadequate for genus-level phylogeny in

Enterobacteriacea (Naum et al., 2008) and for understanding the intergeneric relationships within this family (Adeolu et al., 2016), but it also seems to have some problems at the species level in Xenorhabdus as evidenced here. The presence of recombination along with gene copies that are very different can yield misleading evolutionary information. When working with potential novel species, multiple copies of the gene should be analyzed and this data should be analyzed alongside or combined with multiple protein coding gene sequences.

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Table 4-1. Bacterial isolates used for this study and number of 16S rRNA copies Copies of Bacterial strain Nematode host Geographical origin (Reference) source 16S rRNA X. bovienii (Lengyel et al., 2005) Or1 S. oregonense Oregon, USA K. Nguyena 4 O-1A, O-1B, O-2A, O- 2B X. budapestensis Bico1 S. bicornutum 1 Yugoslavia K. Nguyena 2 B-27, B-29 X. doucetiae (Tailliez et al., 2006) LWD1 S. diaprepesi Florida, USA L. Duncana 2 D-2B, D-2P X. hominickii (Tailliez et al., 2006) Sm1 S. monticolum Korea H. Kayaa 2 M-2A, M-2B X. indica Somvanshi et al., 2006; Nguyen et al., 2007 SabOM S. abbasi Oman Sultanate of Oman K. Nguyena 2 Ab-1 Ab-2 X. koppenhoeferi (Tailliez et al., 2006) AMK1 S. scarabaei AMK001 New Jersey, USA A. Koppenhöfera 3 A-1, A-2 X. kozodoii (Tailliez et al., 2006) Sa1 S. arenarium Russia K. Nguyena 2 X. nematophila (Thomas & Poinar, 1979) A-4, A-5 AN6/1 S. carpocapsae DD136 Virginia, USA R. Akhurstb 1 (Akhurst, 1983; Akhurst & X. poinarii Boemare, 1988) Cu1 S. cubanum Cuba K. Nguyena 1 GK S. glaseri NC North Carolina, USA K. Nguyena 1 FL224 S. glaseri 224 Florida, USA This studya 3 224 4-1, 224 4-2, 224 4-3

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Table 4-1. Continued Copies of Bacterial strain Nematode host Geographical origin (Reference) source 16S rRNA X. szentirmaii (Lengyel et al., 2005; Nguyen et al., 2006) 17C S. rarum LA Louisiana, USA K. Nguyena 2 17-1, 17-2 Xenorhabdus sp. TX26 S. texanum Texas, USA K. Nguyena 2 TX-1, TX-2 Xenorhabdus sp. VN13 Steinernema sp 13 Vietnam K. Nguyena 2 13-1, 13-2 aProvided host nematode bProvided bacteria

Table 4-2. Sequences of primers used to amplify and sequence gene regions Primer Type1 Sequence (5'-3') 16S rRNA FGPS6-63* Amp GGAGAGTTAGATCTTGGCTCAG FGPL132-38* Amp CCGGGGTTTCCCCATTCGG HK1 Seq GGAACGCTGAGACAGGTG HK2 Seq AAGGAGGTGATCCAACCGCAG

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Table 4-3. GenBank accession numbers for additional 16S gene sequences used in this study Bacterial strain Accession No. 16S Complete Genome Xenorhabdus X. beddingii Q58 AY278675 X. bovienii DSM4766 AY278673 X. bovienii SS-2004 FN667741 X. bovienii TB30 DQ208306 X. budapestensis strain DSM16342T AJ810293 X. budapestensis strain CN03 DQ211714 X. cabanillasii strain USTX62 AY521244 X. doucetiae strain FRG30 DQ211702 X. doucetiae strain FRM16 DQ211709 X. ehlersii DSM16337 AJ810294 X. ehlersii KR02 DQ208308 X. griffiniae ID10 DQ211710 X. hominickii KmYb11 AB507815 X. hominickii KR05 DQ205449 X. indica DSM17382 AM040494 X. indica OnIr181 AB507813 X. innexi DSM16336 AJ810292 X. innexi UY61 AY521243 X. ishibashii AB243427 X. ishibashii GDh7 GQ149086 X. japonica DSM16522 DQ202310 X. japonica AB243426 X. khoisanae SF80 JX623966 X. khoisanae strain SF362 JX623978 X. koppenhoeferi UDNJ01 DQ205450 X. kozodoii strain SaV DQ211716 X. kozodoii strain FR48 EU190977 X. magdalenensis IMI 397775 HQ877464 X. mauleonii strain VC01 DQ211715 X. miraniensis strain Q1 DQ211713 X. nematophila ATCC 19061 NC_014228 X. poinarii AZ26 DQ211703 X. poinarii G6 FO704551 X. romanii strain PR06-A DQ211717 X. stockiae strain TH01 DQ202309 X. stockiae SRK7 FJ006728 X. szentirmaii DSM 16338 AJ810295 X. szentirmaii K77 DQ211712 X. vietnamensis VN01 DQ205447 Outgroup Taxa Escherichia coli K12 subMG1655 U00096 Serratia proteamaculans 568 CP000826

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Table 4-4. Similarity values for the 16S rRNA sequence for the two undescribed Xenorhabdus strains to known species and to each other Xenorhabdus 16S rRNA sequence Xenorhabdus 16S X. szentirmaii X. szentirmaii VN13-1 VN13-2 TX26-1 rDNA sequence DSM 16338 K77 X. szentirmaii K77 99.66 VN13 RNA cistron 1 99.07 99.05 VN13 RNA cistron 2 98.68 98.71 99.14 TX26 RNA cistron 1 98.68 98.71 98.29 98.02 TX26 RNA cistron 2 98.74 98.78 98.35 98.09 99.93

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A X. poinarii Cu1 GCGGCAGCGGGAGAAAG CGA—-G CUUUCUUGCCGGCGAGC STEPMATRIX = 4 X. szentirmaii GCGGCAGCGGGAAGAAG CGU—-G CUUCUUUGCCGGCGAGC [ 0 = GA] X. doucetiae D-2B ACGGUAACAGGAAACAG CUU—-G CUGUUUUGCUGACGAGU [ 1 = GU] X. doucetiae D-2P GCGGCAGCGGGAAGUAG CUU—-G CUACUUUGCCGGCGAGC [ 2 = UU] X. cabanillasii GCGGUAACAGGAAAGGG CUUUUG CACUUUUGCUGACGAGC [ 3 = UUUU]

B X. stockiae strain TH01 CCAGCAC AUCAUG GUGGG STEPMATRIX = 7 X. khoisanae SF80 CCAGCAC GUCAUG GUGGG [ 0 = AUCAUG] X. bovienii O-2A CCAGCAC GUGAUG GUGGG [ 1 = GUAAUG] X. bovienii O-2B CCAGCAC GUAAUG GUGGG [ 2 = GUCAUG] X. indica CCAGCAC GUUAUG GUGGG [ 3 = GUGAUG] X. nematophila CCAGCAC UUCGG GUGGG [ 4 = GUUAUG] E. coli J01695 CCAGCGG UCCGG CCGGG [ 5 = UCCGG] [ 6 = UUCGG] C X. cabanillasii GCUUAACC UGCG GGAGGGC STEPMATRIX = 7 X. budapestensis GCUUAACC UGCU GGAGGGC [ 0 = UGCG] X. ehlersii GCUUAACC UGUA GGAGGGC [ 1 = UGCU] X. kozodoii GCUUAACC UUCG GGGGGGC [ 2 = UGUA] X. romanii GCUUAACC UUUAU GGAGGGC [ 3 = UUCG] X. griffiniae GCUUAACC UUUU GGAGGGC [ 4 = UUUAU] X. doucetiae GCUUAACC UUUUU GGAGGGC [ 5 = UUUU] [ 6 = UUUUU]

Figure 4-1. Regions where the 16Sr RNA gene was ambiguously aligned. Stems and loops (center of the aligned sequences) from A) variable region 1, B) variable region 7, and C) variable region 9 of 16S rRNA (Yarza et al., 2014). For maximum parsimony, these regions were coded using Inaase 3.0. For Bayesian analysis, these regions were omitted.

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Figure 4-2. Maximum Parsimony tree based on 16S rRNA sequences. Figure represents one of 12 equally parsimonious trees. Ambiguous regions were coded using INAASE. Bootstrap support values >60 presented. Taxa in blue 16S rRNA amplified for this study.

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Figure 4-3. Bayesian analysis of Xenorhabdus using 16S rRNA sequences, majority rule consensus trees. The analysis was run for 20,000,000 generations, sample frequency = 100 under the GTR+I+G model. The first 25% of trees were discarded as burn-in. Posterior probabilities above 0.60 are presented as percentages.

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Figure 4-4. Split decomposition graph of Xenorhabdus based on 16S rRNA sequences using the uncorrected P characters. The fit score for the graph is 59.93.

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CHAPTER 5 CLOSING REMARKS

The flow of newly described entomopathogenic nematodes is seemingly unending.

Currently, there are at least 17 heterorohabditid species and more than 95 steinernematid species

(Hunt, 2016). Each new nematode presumably possesses a unique relationship with a bacterial partner, providing more opportunity for the use of taxonomic and phylogenic tools and the study of symbiosis and pathogenesis. In addition, three complete genomes and a draft assembly for

Photorhabdus, representing all four species, and several complete genomes and draft assemblies for Xenorhabdus, representing 14 species, are available in GenBank. Many others are in the process of being completed. Together, the availability of the bacterial genomes along with many diverse nematode-bacterium combinations are giving insight into the symbiosis and pathogenicity of the bacteria and aid in the reconstruction of evolutionary and co-evolutionary history.

Though the Photorhabus-Heterorhabditis and Xenorhabdus-Steinernema symbiotic relationships possess many similarities, their association with one another is thought to be one of convergent evolution (Boemare & Akhurst, 2006; Chaston et al., 2011). These are both complex systems in that the bacteria have the ability to inhabit two dissimilar ecological niches: the nematode intestine (nutritionally poor) and the insect cadaver (nutritionally rich). The bacteria are both pathogen and beneficial symbiont. Xenorhabdus and Photorhabdus are phenotypically and genotypically more similar to each other than any other genus, but they possess many differences. In a comparision study of four different genomes, two Photorhabdus and two

Xenorhabdus, Chaston et al. (2011) found many processes to be conserved but also several key features important to pathogenesis and symbiosis to be unique to each genus. This suggested

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that at one point in history, Xenorhabdus and Photorhabdus shared a common ancestor, but they have since diverged.

In the research presented here, the diversity and evolutionary history was examined for

Photorhabdus and Xenorhabdus. Both genera possess a complex history, which requires multiple gene sequences to untangle. There was evidence for recombination in both

Photorhabdus and Xenorhabdus; if it was not the cause of the complexity of the evolutionary relationships among these bacteria, it increased the complexity of the results. Though there is some specificity between heterorhabditid nematodes and their symbiotic bacteria, these nematodes are known to host different symbiotic bacteria (Maneesakorn et al., 2011). Recently, it has been shown that a heterorhabditid nematode can naturally reassociate with a different

Photorhabdus to increase its ecological niche (Maher et al., 2017). This symbiont switching could potentially put two different Photorhabdus species in close proximity where genetic material can be exhanged.

However, the relationship between Xenorhabdus and its nematode host appears to be much more specific. Murfin et al. (2015) found that the fitness in the nematode-bacterium mutualism varied with strains of the same bacterial species and that the nematodes with their native strain or closely related strain of bacterium faired better than with a more divergent strain.

Xenorhabdus produce many chemicals including xenocoumacins (McInerney et al., 1991) and bacteriocins (Boemare et al., 1992; Singh & Banjeree, 2008; Morales-Soto & Forst, 2011), which cause the death of bacteria from other genera, other species of Xenorhabdus and even or different strains within a species of Xenorhabdus. With Xenorhabdus having this kind of arsenal, it is difficult to imagine how they can undergo recombination. Although not common, bacteria insensitivity to bacteriocins in Xenorhabdus has been recorded (Siccard et al., 2005). In

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other systems, bacterial recombination does occur among highly competitive species. Timilsina et al. (2015) reported bacterial recombination between Xanthomonas euvesicatoria and X. perforans, which are known to produce bacteriocins to outcompete similar strains of bacteria.

The data presented here and by others (Sergeant et al., 2006; Tailliez et al., 2010) does indicate homologous recombination among these highly competitive species.

It is difficult to know what genetic information is recently derived and what information is truly ancestral. In the first study presented here involving Photorhabdus, only two protein- coding genes were used, gyrB and rpoB, both of wich appeared to have undergone recombination or some other evolutionary event that could potentially give artificial phylogenetic data. Because the data suggested that recombination occurred in different regions of the gene sequence for different species, finding a universal fragment free of recombination and still evolutionarly discriminating is difficult. In order to tease out these phylogentic relationships, it is necessary to use sequence data from multiple genes. With more data, it is hopeful that misleading phylogenetic signals will be dampened.

For constructing the Xenorhabdus phylogeny presented in chapter 3, four protein coding gene sequences were used, the two aforementioned genes and groEL and recA. Again, there was statistical evidence for recombination within rpoB, groEL and potentially gyrB. Because there was still some discrepancy between the trees presented here and other published Xenorohabdus trees (Ferreira, et al., 2014; Tailliez; et al., 2012) where five genes were used, it is important to use sequences from more genes.

The list of accepted Xenorhabdus species is growing. Currently there are 24 described species of Xenorhabdus and two more have recently been proposed, X. thuongxuanensis and X. eapokensis (Kämpfer et al., 2017). Though there are Xenorhabdus species that associate with

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more than one steinernamtid nematode (e.g., X. poinarii and X. bovienii), with so many described nematode species, there is potential for even more species richness among Xenorhabdus. In the studies presented here in chapters 3 and 4, there are two undescribed Xenorhabdus isolates that were included in the study. The 16S rRNA gene sequence data suggested that they could be strains of X. szentirmaii. Accordingly, with the protein coding sequence data, the Xenorhabdus sp. isolated from S. texanum could very well be. However, the strain isolated from the undescribed nematode from Vietnam, could be a new species. More studies would have to be conducted to determine the species designation for both of these strains. Within the genus

Photorhabdus, there are only four species, but each has multiple subspecies except for the most recently described species, P. heterorhabditis. The work conducted here did not suggest any novel species or subspecies. In chapter 2 is presented the first record of P. heterorhabditis in the

U.S. It is the bacterial symbiont found associated with H. zealandica from Forida and South

Carolina.

The utility of rep-PCR in discriminating species (Xenorhabdus and Photorhabdus) and subspecies of Photorhabdus was demonstrated in these studies. It could be used alongside multiple gene analyses to confirm species designations. With the increasing bank of genomic information, more data can be easily acquired to tease out these evolutionary relationships.

Gathering more information about the bacteria and their host nematodes will be crucial in developing a better understanding of the system, its richness and its applications for biological use.

168

APPENDIX A rRNA SEQUENCES

X. poinarii strain FL224 4-3, 16S ribosomal RNA gene, partial sequence; 16S-23S ribosomal RNA intergenic spacer, complete sequence; and 23S ribosomal RNA gene, partial sequence. >FL224_4-3 GGAGAGUUAAUCUUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAGCGGCAGCGGGAGAAA GCGUGCUUUCUUGCCGGCGAGCGGCGGACGGGUGAGUAAUGUCUGGGGAUCUGCCCGAUGGCGGGGGAUAACCAC UGGAAACGGUGGCUAAUACCGCAUAAUCUCUGUGGAGCAAAGUGGGGGACCUUCGGGCCUCACGCCAUCGGAUGA ACCCAGAUGGGAUUAGCUAGUAGGUGGGGUAAAGGCUCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGA CCAGCCACACUGGGACUGAGACACGGCCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGC AAGCCUGAUGCAGCCAUGCCGCGUGUAUGAAGAAGGCCUUCGGGUUGUAAAGUACUUUCAGCGGGGAGGAAGGCG GCAGCCUGAAUAAGGUUGGCGUUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGU AAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCAAUUAAGUUAGAUGUG AAAUCCCCGGGCUUAACCUGGGAACGGCAUCUAAGACUGGUUGGCUAGAGUCUCGUAGAGGGGGGUAGAAUUCCA CGUGUAGCGGUGAAAUGCGUAGAGAUGUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACGAAGACUGACGC UCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUUGGA GGUUGUGGCCUAGAGCUGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGUACGGCCGCAAGGUUA AAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCU UACCUACUCUUGACAUCCACGGAAUUUUGCAGAGAUGCGAAAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAU GGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAG CACGUAAUGGUGGGAACUCAAGGGAGACUGCCGGUGAUAAACCGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAU GGCCCUUACGAGUAGGGCUACACACGUGCUACAAUGGCAGAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGA CCUCAUAAAGUCUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUA GAUCAGAAUGCUACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGGGUGGGUUGCAAA AGAAGUAGGUAGCUUAACCUUCGGGGGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGG UAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUUAAACCGACGAUAGGUUAUUGUUGCAGUGCUCACACAGAU UGUCUGAUGAAAAUGAAGAAGAAGCAGCGCGUCUGCGAAGAAGACUGAUGUCCCCUUCGUCUAGAGGCCUAGGAC ACCGCCCUUUCACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGCCACGCUGCGGGGAACGGGUGAAAGGCG UAACCAACUGUAUUUUUAAGUGGGCUGGUGACAGCCGAUUUACAAAUCAUGCUCUUUAACAAUCUGGAACAAGCU GAAAAUUUGAAACACUCAGUGCUGUCAAGACAGUAUUGAGGCGUCUCUCAACCCGCACUCCGAAGACACCUUCGG GUUGUGAGGUUAAGCGAAUAAGCGUACACGGUGGAUGCCUAGGCAGUCAGAGGCGAUGAAGGACGUGCUAAUCUG CGAUAAGCGCCGGUAAGGUGAUAUGAACCGUGAUAGCCGGCGAUGUCCGAAUGGGGAAACCCGG

Xenorgabdus sp. strain TX-26 TX-2, 16S ribosomal RNA gene, partial sequence; 16S-23S ribosomal RNA intergenic spacer, complete sequence; and 23S ribosomal RNA gene, partial sequence. >TX26_TX-2 GGAAGUUAGAUCUUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAGCGGCAGCGGGAAGAA GCGUGCUUCUUUGCCGGCGAGCGGCGGACGGGUGAGUAAUGUCUGGGGAUCUGCCUGAUGGAGGGGGAUAACCAC UGGAAACGGUGGCUAAUACCGCAUGACCUCUGAGGAGCAAAGUGGGGGACCUUCGGGCCUCACGCCAUCGGAUGA ACCCAGAUGGGAUUAGCUAGUAGGCGGGGUAAUGGCCCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGA CCAGCCACACUGGGACUGAGACACGGCCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGC AAGCCUGAUGCAGCCAUGCCGCGUGUAUGAAGAAGGCCUUCGGGUUGUAAAGUACUUUCAGCGGGGAGGAAGGCG UGCGUCCGAAUACGGCGCACGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGU AAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCAAUUAAGUUAGAUGUG AAAUCCCCGGGCUUAACCUGGGAACGGCAUCUAAGACUGGUUGGCUAGAGUCUCGUAGAGGGGGGUAGAAUUCCA CGUGUAGCGGUGAAAUGCGUAGAGAUGUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACGAAGACUGACGC UCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUUGGA GGCUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGUACGGCCGCAAGGUUA AAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCU UACCUACUCUUGACAUCCACAGAAUCUGGCAGAGAUGCCGGAGUGCCUUCGGGAACUGUGAGACAGGUGCUGCAU GGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAG CACGUGAUGGUGGGAACUCAAGGGAGACUGCCGGUGAUAAACCGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAU GGCCCUUACGAGUAGGGCUACACACGUGCUACAAUGGCAGAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGA

169

ACUCAUAAAGUCUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUA GAUCAGAAUGCUACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAA AGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGG UAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUUACCGAACGGUAGUCAUUCCUGUGCAGUGCCCACACAGAU UGUCUGAUGAAAGAAAGAAUGAGCAGCGUCUGCGAAGAAGACUUGAAGUCCCCUUCGUCUAGAGGCCUAGGACAC CGCCCUUUCACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGCCACUUUGCUCGCGGGACUGGGUGAAAGCC GGUGCCAACCGAUAUUGUUAAGCGUGACUUGCGAGUCAGUUUAGCAAUGUUUGCUCUUUAACAAUCUGGAACAAG CUGAAAAUUUGAAACACUCAGUACUGUCACACGGUACUGAGAAGUCUCUCAAAAACUCCGAUCCGAAGACACCUU CGGGUUGUGAGGUUAAGCGACUAAGCGUACACGGUGGAUGCCUAGGCAGUCAGAGGCGAUGAAGGGCGUGCUAAU CUGCGAUAAGCGACGGCAAGGUGAUAUGAACCGCAACACCCGUCGAUACCCGAAUGGGGAAACCCGG

170

APPENDIX B FATTY ACID METHYL ESTER COMPOSITION

Table B-1. X. nematophila, ATCC 39497 RT Response Ar/Ht RFact ECL Peak Name Percent 1.655 3.898E+8 0.025 ---- 7.055 SOLVENT PEAK ---- 2.956 674 0.028 ---- 10.074 ---- 3.457 227 0.025 1.138 10.914 Sum In Feature 2 0.11 4.315 4086 0.028 1.069 12.001 12:0 1.88 4.944 2045 0.031 1.037 12.612 13:0 ISO 0.91 6.361 1118 0.034 ---- 13.812 ---- 6.598 23286 0.038 0.981 14.000 14:0 9.84 7.190 385 0.035 0.967 14.412 15:1 ISO F 0.16 7.318 2152 0.042 0.965 14.501 unknown 14.502 0.89 7.492 7002 0.042 0.961 14.622 15:0 ISO 2.90 8.039 3391 0.037 0.952 15.002 15:0 ---- 8.321 1602 0.045 ---- 15.180 ---- 8.467 437 0.030 ---- 15.272 ---- 8.805 20912 0.040 0.940 15.486 Sum In Feature 2 8.47 9.327 59793 0.040 0.934 15.816 Sum In Feature 3 24.05 9.622 74492 0.042 0.930 16.002 16:0 29.86 9.840 5173 0.042 0.928 16.133 15:0 ISO 3OH 2.07 10.309 505 0.037 0.923 16.414 ISO 17:1 w9c 0.20 10.467 699 0.046 0.922 16.509 15:0 3OH 0.28 10.668 1084 0.038 0.920 16.629 17:0 ISO 0.43 11.096 9554 0.045 0.917 16.886 17:0 CYCLO 3.77 11.583 693 0.042 ---- 17.173 ---- 11.824 1375 0.045 ---- 17.314 ---- 12.173 5677 0.043 0.910 17.517 16:0 3OH 2.22 12.693 28568 0.044 0.907 17.820 18:1 w7c 11.16 13.007 420 0.037 0.905 18.003 18:0 0.16 14.545 761 0.037 0.898 18.900 19:0 CYCLO w8c 0.29 15.333 862 0.043 0.895 19.362 19:0 10 methyl 0.33 ---- 21138 ------Summed Feature 2 8.58 ------59793 ------Summed Feature 3 24.05

171

Table B-2. X. szentirmaii, 17C RT Response Ar/Ht RFact ECL Peak Name Percent 1.655 3.84E+8 0.025 ---- 7.057 SOLVENT PEAK ---- 2.153 853 0.021 ---- 8.220 ---- 2.192 3022 0.021 ---- 8.310 ---- 2.370 552 0.022 ---- 8.727 ---- 2.407 1118 0.023 ---- 8.814 ---- 2.912 411 0.023 1.209 9.999 10:0 0.23 4.315 8808 0.028 1.069 12.001 12:0 4.37 4.945 1382 0.031 1.037 12.613 13:0 ISO 0.66 5.346 773 0.033 1.019 13.002 13:0 0.37 5.912 1769 0.034 1.000 13.453 12:0 3OH 0.82 6.361 1265 0.034 ---- 13.812 ---- 6.598 21155 0.036 0.981 14.001 14:0 9.62 6.753 437 0.035 0.977 14.109 13:0 ISO 3OH 0.20 7.318 2119 0.054 0.965 14.501 unknown 14.502 0.95 7.493 2436 0.038 0.961 14.622 15:0 ISO 1.09 7.708 1161 0.044 ---- 14.772 ---- 8.040 4294 0.036 0.952 15.003 15:0 ---- 8.323 429 0.034 ---- 15.181 ---- 8.466 634 0.040 ---- 15.271 ---- 8.805 14887 0.041 0.940 15.486 Sum In Feature 2 6.49 9.326 20774 0.041 0.934 15.814 Sum In Feature 3 8.99 9.622 64616 0.040 0.930 16.001 16:0 27.87 9.772 1371 0.052 ---- 16.091 ---- 9.998 525 0.043 0.927 16.226 15:0 2OH 0.23 10.456 609 0.041 0.922 16.501 15:0 3OH 0.26 11.097 42298 0.043 0.917 16.885 17:0 CYCLO 17.98 11.288 712 0.034 0.915 16.999 17:0 0.30 11.822 1729 0.043 ---- 17.310 ---- 12.173 2277 0.044 0.910 17.515 16:0 3OH 0.96 12.582 1144 0.043 ---- 17.753 ---- 12.693 35867 0.044 0.907 17.818 18:1 w7c 15.07 13.005 960 0.044 0.905 18.000 18:0 0.40 13.761 1543 0.048 ---- 18.440 ---- 14.109 990 0.050 0.900 18.643 19:0 ISO 0.41 14.545 1637 0.039 0.898 18.896 19:0 CYCLO w8c 0.68 14.665 714 0.040 ---- 18.966 ---- 15.169 1996 0.051 0.896 19.261 18:0 2OH 0.83 15.330 811 0.042 0.895 19.355 19:0 10 methyl 0.34 ---- 14887 ------Summed Feature 2 6.49 ------20774 ------Summed Feature 3 8.99

172

Table B-3. X. koppenhoeferi, AMK001 RT Response Ar/Ht RFact ECL Peak Name Percent 1.656 3.809E+8 0.025 ---- 7.056 SOLVENT PEAK ---- 4.315 3179 0.028 1.069 12.001 12:0 2.94 4.944 586 0.031 1.037 12.612 13:0 ISO 0.53 5.702 338 0.033 1.007 13.285 12:1 3OH 0.29 5.912 1591 0.034 1.000 13.453 12:0 3OH 1.38 6.357 342 0.033 ---- 13.808 ---- 6.599 12721 0.036 0.981 14.001 14:0 10.79 7.319 933 0.038 0.965 14.502 unknown 14.502 0.78 7.492 921 0.036 0.961 14.622 15:0 ISO 0.77 8.040 935 0.034 0.952 15.003 15:0 ---- 8.468 495 0.039 ---- 15.273 ---- 8.806 7558 0.040 0.940 15.486 Sum In Feature 2 6.15 9.327 37695 0.041 0.934 15.816 Sum In Feature 3 30.43 9.622 34781 0.041 0.930 16.002 16:0 27.98 10.671 372 0.035 0.920 16.631 17:0 ISO 0.30 11.097 9010 0.044 0.917 16.886 17:0 CYCLO 7.14 12.584 513 0.041 ---- 17.757 ---- 12.693 13431 0.043 0.907 17.820 18:1 w7c 10.53 13.761 727 0.041 ---- 18.443 ------7558 ------Summed Feature 2 6.15 ------37695 ------Summed Feature 3 30.43

Table B-4. X. koppenhoeferi, AMK002 RT Response Ar/Ht RFact ECL Peak Name Percent 1.656 3.786E+8 0.025 ---- 7.055 SOLVENT PEAK ---- 4.316 5454 0.028 1.069 12.000 12:0 3.22 5.704 532 0.032 1.007 13.286 12:1 3OH 0.30 5.912 2482 0.034 1.000 13.452 12:0 3OH 1.37 6.362 454 0.033 ---- 13.811 ---- 6.599 15978 0.037 0.981 14.000 14:0 8.64 7.319 1477 0.039 0.965 14.501 unknown 14.502 0.79 8.041 652 0.034 0.952 15.003 15:0 ---- 8.323 597 0.030 ---- 15.181 ---- 8.467 873 0.036 ---- 15.271 ---- 8.807 11083 0.040 0.940 15.486 Sum In Feature 2 5.75 9.328 55956 0.041 0.934 15.815 Sum In Feature 3 28.82 9.475 679 0.040 0.932 15.908 16:1 w5c 0.35 9.623 53187 0.040 0.930 16.001 16:0 27.30 11.098 12817 0.044 0.917 16.886 17:0 CYCLO 6.48 12.583 468 0.039 ---- 17.754 ---- 12.694 33463 0.042 0.907 17.819 18:1 w7c 16.74 13.006 493 0.039 0.905 18.001 18:0 0.25 13.762 622 0.045 ---- 18.441 ----

173

Table B-5. X. bovienii, XbINT (S. intermedium) RT Response Ar/Ht RFact ECL Peak Name Percent 1.656 3.814E+8 0.025 ---- 7.055 SOLVENT PEAK ---- 2.914 403 0.025 1.209 10.000 10:0 0.21 2.958 325 0.026 ---- 10.074 ---- 4.317 12692 0.029 1.069 12.001 12:0 5.78 4.947 470 0.030 1.037 12.612 13:0 ISO 0.21 5.088 356 0.031 ---- 12.749 ---- 5.350 261 0.026 1.019 13.002 13:0 0.11 6.364 770 0.031 ---- 13.812 ---- 6.601 12702 0.040 0.981 14.000 14:0 5.31 7.321 2164 0.042 0.965 14.501 unknown 14.502 0.89 7.493 457 0.030 0.961 14.620 15:0 ISO 0.19 8.042 2988 0.039 0.952 15.002 15:0 ---- 8.324 1117 0.042 ---- 15.180 ---- 8.468 1401 0.040 ---- 15.270 ---- 8.809 20518 0.041 0.940 15.486 Sum In Feature 2 8.22 9.329 18516 0.041 0.934 15.814 Sum In Feature 3 7.36 9.479 455 0.035 0.932 15.909 16:1 w5c 0.18 9.626 85096 0.042 0.930 16.002 16:0 33.73 9.781 2446 0.064 ---- 16.094 ---- 11.101 54892 0.044 0.917 16.886 17:0 CYCLO 21.44 11.293 798 0.035 0.915 17.001 17:0 0.31 11.828 1797 0.044 ---- 17.312 ---- 12.230 3079 0.046 ---- 17.547 ---- 12.586 1517 0.040 ---- 17.754 ---- 12.696 17446 0.044 0.907 17.818 18:1 w7c 6.74 13.005 1213 0.043 0.905 17.999 18:0 0.47 13.761 2007 0.053 ---- 18.439 ---- 14.109 1240 0.047 0.900 18.641 19:0 ISO 0.48 14.547 20920 0.045 0.898 18.897 19:0 CYCLO w8c 8.00 14.664 787 0.040 ---- 18.964 ---- 15.368 986 0.046 0.895 19.377 19:0 10 methyl 0.38 ---- 20518 ------Summed Feature 2 8.22 ------18516 ------Summed Feature 3 7.36

174

Table B-6. P. temperata (H. megidis) RT Response Ar/Ht RFact ECL Peak Name Percent 1.657 3.806E+8 0.025 ---- 7.055 SOLVENT PEAK ---- 4.317 2323 0.028 1.064 12.000 12:0 2.18 4.946 2108 0.029 1.033 12.611 13:0 ISO 1.92 5.350 280 0.033 1.015 13.003 13:0 0.25 6.362 573 0.037 ---- 13.811 ---- 6.600 8126 0.038 0.978 14.001 14:0 7.00 7.191 1184 0.037 0.966 14.411 15:1 ISO F 1.01 7.319 893 0.045 0.963 14.500 unknown 14.502 0.76 7.494 12014 0.036 0.960 14.622 15:0 ISO 10.16 7.623 534 0.036 0.957 14.711 15:0 ANTEISO 0.45 8.041 2408 0.039 0.950 15.002 15:0 ---- 8.807 5845 0.039 0.940 15.486 Sum In Feature 2 4.84 9.032 540 0.035 0.937 15.628 16:0 ISO 0.45 9.328 20886 0.041 0.934 15.815 Sum In Feature 3 17.18 9.623 27319 0.040 0.931 16.001 16:0 22.40 9.842 2498 0.040 0.929 16.133 15:0 ISO 3OH 2.04 10.311 6674 0.041 0.925 16.414 ISO 17:1 w9c 5.44 10.673 7403 0.041 0.922 16.630 17:0 ISO 6.01 10.827 608 0.034 0.921 16.723 17:0 ANTEISO 0.49 11.098 3968 0.044 0.919 16.885 17:0 CYCLO 3.21 11.292 673 0.039 0.918 17.002 17:0 0.54 12.696 17046 0.046 0.910 17.820 18:1 w7c 13.67 ---- 5845 ------Summed Feature 2 4.84 ------20886 ------Summed Feature 3 17.18

Table B-7. P. heterorhabditis, ENY25 RT Response Ar/Ht RFact ECL Peak Name Percent 1.654 3.779E+8 0.026 ---- 7.060 SOLVENT PEAK ---- 4.319 438 0.034 ---- 11.972 ---- 8.862 449 0.039 0.939 15.483 Sum In Feature 2 0.85 9.384 748 0.036 0.934 15.812 Sum In Feature 3 1.42 9.679 2403 0.042 0.932 15.998 16:0 4.54 11.356 510 0.039 0.923 17.003 17:0 0.95 12.761 40409 0.045 0.919 17.821 18:1 w7c 75.20 13.070 2349 0.043 0.919 18.001 18:0 4.37 14.614 2506 0.046 0.916 18.900 19:0 CYCLO w8c 4.65 14.928 3106 0.048 0.915 19.084 18:1 2OH 5.75 15.723 1229 0.049 0.914 19.551 18:0 3OH 2.27 ---- 449 ------Summed Feature 2 0.85 ------748 ------Summed Feature 3 1.42

175

Table B-8. P. luminescens subsp. laumondii, (H. bacteriophora, HP88) RT Response Ar/Ht RFact ECL Peak Name Percent 1.657 3.786E+8 0.025 ---- 7.055 SOLVENT PEAK ---- 3.275 218 0.022 1.155 10.606 11:0 ISO 0.05 4.317 8533 0.028 1.064 12.001 12:0 1.96 4.946 6136 0.030 1.033 12.612 13:0 ISO 1.37 5.350 405 0.030 1.015 13.003 13:0 0.09 5.913 1310 0.033 0.997 13.453 12:0 3OH 0.28 6.119 1047 0.034 0.991 13.617 14:0 ISO 0.22 6.364 1915 0.036 ---- 13.812 ---- 6.601 24833 0.037 0.978 14.001 14:0 5.23 7.191 3240 0.035 0.966 14.411 15:1 ISO F 0.67 7.319 2389 0.045 0.963 14.500 unknown 14.502 0.50 7.496 52231 0.037 0.960 14.623 15:0 ISO 10.81 7.624 2813 0.036 0.957 14.711 15:0 ANTEISO 0.58 8.041 3757 0.037 0.950 15.001 15:0 ---- 8.230 1477 0.039 0.948 15.121 14:0 ISO 3OH 0.30 8.322 775 0.041 ---- 15.179 ---- 8.808 18725 0.041 0.940 15.486 Sum In Feature 2 3.79 9.031 5095 0.041 0.937 15.627 16:0 ISO 1.03 9.331 99773 0.040 0.934 15.816 Sum In Feature 3 20.08 9.474 930 0.045 0.932 15.906 16:1 w5c 0.19 9.625 96435 0.039 0.931 16.002 16:0 19.35 9.842 7742 0.041 0.929 16.132 15:0 ISO 3OH 1.55 10.312 21407 0.042 0.925 16.414 ISO 17:1 w9c 4.27 10.674 45587 0.043 0.922 16.631 17:0 ISO 9.06 10.826 4024 0.044 0.921 16.723 17:0 ANTEISO 0.80 10.941 715 0.038 0.920 16.791 17:1 w8c 0.14 11.098 2012 0.060 0.919 16.885 17:0 CYCLO 0.40 11.292 1638 0.044 0.918 17.002 17:0 0.32 12.698 85021 0.044 0.910 17.822 18:1 w7c 16.68 13.004 1406 0.043 0.909 18.000 18:0 0.28 13.727 619 0.048 ---- 18.421 ------18725 ------Summed Feature 2 3.79 ------99773 ------Summed Feature 3 20.08

176

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BIOGRAPHICAL SKETCH

Heather Smith Koppenhöfer was born in Springfield, New York, to Stephen and Linda

Smith. She earned her Bachelor of Science degree in molecular biology and microbiology from the University of Central Florida, Orlando, Florida, in 1994 and went on to earn a Master of

Science degree from the same department. Heather worked for the University of Florida Gulf

Coast Research and Education Center in the laboratory of Dr. David Schuster. Through his encouragement she returned to school to pursue her PhD degree at the University of Florida. In

1999, Heather was awarded an alumni fellowship and began her program in the department of

Entomology and Nematology investigating the diversity of bacteria symbiotically associated with entomopathogenic nematodes under the supervision of Drs. Byron J. Adams and Jeffrey B.

Jones. Due to unforeseen circumstances, Heather had to leave her program. Several years later with the encourgagement of her professors, she returned to complete her research under the supervision of Drs. Jeffrey B. Jones and James E. Maruniak.

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