HOST SPECIFICITY AND REGIONAL ENDEMICITY IN SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM, DINOPHYTA) ASSOCIATED WITH SEA ANEMENOES IN THE GENUS AIPTASIA Except where reference is made to the work of others, the work describe in this thesis is my own or was one in collaboration with my advisory committee. This thesis does not include proprietary or classified information. _______________________________ Yu Xiang Certificate of Approval: _______________________________ Kenneth M. Halanych Associate Professor Biological Sciences _______________________________ Stephen C. Kempf Associate Professor Biological Sciences _______________________________ Scott R. Santos, Chair Assistant Professor Biological Sciences _______________________________ Leslie R. Goertzen Assistant Professor Biological Sciences _______________________________ George T. Flowers Dean Graduate School HOST SPECIFICITY AND REGIONAL ENDEMICITY IN SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM, DINOPHYTA) ASSOCIATED WITH SEA ANEMENOES IN THE GENUS AIPTASIA Yu Xiang A Thesis Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Master of Science Auburn, Alabama May 9, 2009 iii HOST SPECIFICITY AND REGIONAL ENDEMICITY IN SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM, DINOPHYTA) ASSOCIATED WITH SEA ANEMENOES IN THE GENUS AIPTASIA Yu Xiang Permission is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. ______________________________ Signature of Author ______________________________ Date of Graduation iv THESIS ABSTRACT HOST SPECIFICITY AND REGIONAL ENDEMICITY IN SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM, DINOPHYTA) ASSOCIATED WITH SEA ANEMENOES IN THE GENUS AIPTASIA Yu Xiang Master of Sciences, May 9, 2009 (B.S., Qufu Normal University, 2001) 101 Typed Pages Directed by Scott R. Santos Recent investigations of coral reef biology have focused on the global biogeography and host specificity of Symbiodinium, a diverse group of dinoflagellates that symbiotically associate with many marine organisms, including reef-building corals. Despite this, few studies have investigated the genetic structure of Symbiodinium at the population level. One suitable system to investigate Symbiodinium population genetics of a single host across a global range is the facultatively symbiotic anemone Aiptasia. In order to determined the specificity and population genetic structure of Symbiodinium communities associated with Aiptasia spp., 356 anemones were sampled from 18 locations throughout the world. Symbiodinium diversity was assessed using a variety of v molecular markers that measure diversity from the level of sub-generic clades to populations, including restriction fragment length polymorphisms (RFLPs) of 18S-rDNA, denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer 2 (ITS2) rDNA, flanking region sequences of two microsatellite loci, and allelic variation at six microsatellite loci specific for Symbiodinium Clade B. These data revealed that, with the exception of individuals from the Florida Keys, a single phylotype of Symbiodinium clade B (ITS2 ?type? B1) associates with Aiptasia throughout the world. Furthermore, strong population structure was detected across local, regional, and global geographic scales, suggesting limited gene flow among most Symbiodinium populations. The high genetic structure of Symbiodinium populations and the association with one particular symbiont lineage across large geographic scales suggests strong regional endemism and the existence of specificity in Aiptasia-Symbiodinium symbioses. This work represents a contribution towards our understanding of the ecology and evolution of cnidarian-Symbiodinium endosymbioses. vi ACKNOWLEDGEMENTS The author would like to thank Dr. Scott R. Santos. Without Dr. Santos? support and professional guidance, this thesis would not have been completed. His scientific spirits and precise research methodologies will be beneficial in my future career. I would also like to express deep thanks to Dr. Ken Halanych for his helpful suggestions and support when utilizing facilities in his lab as well as Dr. Leslie Goertzen and Dr. Stephen Kempf for advice during the completion of the thesis. Additional individuals who contributed to helping me complete this thesis and that I would like to thank include: Dr. Dan Thornhill, who gave numerous suggestions on this thesis and invaluable revisions on the manuscript; Dr. Todd C. LaJeunesse and other collaborators who provided Aiptasia holobiont samples from various locations around the world; Dustin W. Kemp, Dr. William K. Fitt, and Dr. Gregory W. Schmidt for the ITS2-DGGE profiling of samples included in this work. Thanks to all my friends, Rebecca Hunter, Mark Liu and Ke Jiang, for their help when studying together. This work was partly supported by the Alabama Commission on Higher Education Graduate Research Scholar's Program through the Auburn University CMB Program, and partly supported by the Graduate School, Auburn University. Lastly, I would greatly thank my family, my wife Min Zhong for their support, encouragement and love, and thank my daughter, Grace Xiang, for being the best new-born, which are always the power for me to move forward. vii Style manual or journal used: Molecular Ecology Computer software used: CLUSTAL_X, FSTAT V2.9.3.2, GENETIC DATA ANALYSIS, Microsoft Excel 2003, Microsoft Word 2003, MODELTEST v3.7, PAUP4.0b10, SEQUENCHER 4.7, TOOL FOR POPULATION GENETIC ANALYSIS (TFPGA) V1.3, TABLE OF CONTENTS LIST OF TABLES...............................................................................................................x LIST OF FIGURES.......................................................................................................... xii CHAPTER 1: LITERATURE REVIEW.............................................................................1 INTRODUCTION...................................................................................................2 THE SPECIFICITY OF THE SYMBIOSIS............................................................4 ESTABLISHMENT AND MAINTENANCE OF SYMBIOTIC RELATIONSHIP.....................................................................................................7 METABOLIC INTERCHANGES IN SYMBIOSIS.............................................10 BLEACHING: BREAKDOWN OF SYMBIOSIS................................................12 TEMPERATE VS. TROPICAL SYMBIOSES.....................................................14 SUMMARY?.......................................................................................................16 LITERATURE CITED..........................................................................................17 CHAPTER 2: HOST SPECIFICITY AND REGIONAL ENDEMICITY IN SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM, DINOPHYTA) ASSOCIATED WITH SEA ANEMENOES IN THE GENUS AIPTASIA.....................................................................29 INTRODUCTION.................................................................................................30 MATERIALS AND METHODS...........................................................................34 RESULTS..............................................................................................................41 DISCUSSION........................................................................................................47 viii CONCLUSION......................................................................................................54 LITERATURE CITED..........................................................................................55 SUMMARY.......................................................................................................................65 TABLES............................................................................................................................67 FIGURES...........................................................................................................................76 APPENDIX TABLE..........................................................................................................80 ix x LIST OF TABLES 2.1. Information on Symbiodinium cultures used in analyses of the six microsatellites specific to Clade B. The host from which the culture was isolated, location of isolation, and microsatellite analysis results are included.................................................................37 2.2. GenBank Accession numbers for the flanking regions of two microsatellite loci in Symbiodinium populations from Aiptasia spp. and algal cultures.....................................38 2.3. Sequence information, annealing temperatures and MgCl 2 concentrations of Symbiodinium Clade B microsatellite primers used in this study......................................39 2.4. Clades (based on 18S-rDNA RFLP) and ITS2 ?types? (based on ITS2 DGGE) of Symbiodinium associated with Aiptasia spp. anemones from throughout the world..................................................................................................................................40 2.5. Genotypic frequencies of six microsatellite loci in Symbiodinium Clade B associated with Aiptasia spp. from throughout the world...................................................................41 2.6. Heterozygosity for six microsatellite loci in Symbiodinium Clade B from Aiptasia spp. across the world..........................................................................................................42 2.7. F ST and ST ? (population differentiation) estimates of Symbiodinium Clade B from Aiptasia spp. across the global range based on six microsatellite loci..............................43 2.8. Symbiodinium Clade B pairwise tests of symbiont population differentiation for Aiptasia spp. at 17 sites containing Symbiodinium Clade B in the world (site abbreviations, see table 1; NS not significant; NA not available; * P<0.05) .................. 44 xi xii LIST OF FIGURES 2.1. Locations of the Aiptasia spp. populations collected from eight major geographic localities across the global range of this host. Geographic localities denoted by two-letter abbreviations as follows: HI = Hawai?ian islands; MX = Mexico; FL = Florida Keys; BR = Bermuda; RS = Red Sea; TH = Thailand; JP = Japan and AU = Australia...................46 2.2. Inferred unrooted phylogenetic relationships between Symbiodinium Clade B based on concatenated flanking regions of microsatellite loci CA4.86 and Si15. Maximum likelihood (ML) tree (?ln L = 622.68). Numbers before and after slashes are support values based on 1000 bootstrap replications (Parsimony/Likelihood respectively). For locations of Aiptasia spp. and cultures, original host name and sample locations of cultures see table 2.............................................................................................................47 2.3. Dendrogram by unweighted pair group method using arithmetic averages (UPGMA) depicting relationships between Symbiodinium Clade B populations of Aiptasia spp. at 17 geographic localities across the global range of the host...................................................48 1 CHAPTER 1 LITERATURE REVIEW 2 I. INTRODUCTION Dinoflagellates in the genus Symbiodinium (Freudenthal 1962) are single-celled eukaryotic microorganism. Members of the genus exclusively form endosymbiotic relationships with other protists or invertebrates such as reef corals, where they acquire access to inorganic carbon, nitrogen and phosphorous from the host. Those chemical elements are then fixed into organic compounds by photosynthesis in the chloroplast of Symbiodinium. In exchange, Symbiodinium pass over 90% of the newly fixed carbon to their host (Muscatine et al. 1981). Thus, this endosymbiosis is regarded as one of the most prominent intracellular association in the sea and underpins the remarkable productivity and biodiversity of coral reef ecosystems worldwide. However, human-associated events such as global warming events are having significant impacts on coral reef ecosystems. One of the most common and visible threats to corals is referred to as ?bleaching?, which is the loss of Symbiodinium or pigments from the algae (Brown 1987; Glynn 1991). In many cases, if a bleached host does not reacquire its symbionts, death may occur. Thus, protection of coral reef ecosystems from such threats is becoming a topic of interest in conservation biology. In order to better understand the basic biology of this endosymbiosis, there is a need to find a model system of cnidarian-algae symbiosis. While Symbiodinium forms relationships with various Cnidaria, Mollusks, Porifera and Protists, sea anemones in the genus Aiptasia has been proposed as the model organism to reveal genetic and physiological characteristics 3 of endosymbioses due to their ease of isolation in the field and culture in the laboratory (Weis et al. 2008). While the genetic diversity of Symbiodinium has been well-studied (Coffroth & Santos 2005), little work has focused on the genetics of Aiptasia. One possible reason for this focus is that mitochondrial DNA, which serves as a popular molecular marker for revealing genetic diversity in animals, has a relatively slow evolution rate in Anthozoas, which hinders its use in such a context (Shearer et al. 2005). This review aims to summarize research on Aiptasia-Symbiodinium symbioses over the past decades. In particularly, a brief research background on the genetic diversity of Symbiodinium was introduced. Then, this review extends from how specific and flexible is the symbiosis between Aiptasia and Symbiodinium; to how the endosymbiosis is established, maintained. Additionally, hypotheses concerning bleaching were stated. Finally, this review attempts to provide suggestions for future research on Aiptasia-Symbiodinium endosymbiosis. 4 II. THE SPECIFICITY OF THE SYMBIOSIS Genetic diversity of Symbiodinium and Aiptasia: Although Symbiodinium was once regarded as a monotypic genus (Taylor 1974), current understandings come to an agreement that Symbiodinium is a heterogeneous group. So far, eight clades of Symbiodinium (Clade A through H) have been formally described with molecular approaches such as Restriction Fragment Length Polymorphism (RFLP) analysis of small subunit of nuclear ribosome DNA (SSrDNA) (Rowan & Powers 1991; Carlos et al. 1999; LaJeunesse & Trench 2000; LaJeunesse 2001; Pochon et al. 2001; reviewed by Coffroth & Santos 2005), with more than one possible species or strain existing in each clade. Relationships between these Symbiodinium clades have been inferred from partial large subunit rDNA (LSU rDNA), internal transcribed spacer region 2 (ITS 2) region (Pochon et al. 2004), mitochondrial genes (Takabayashi et al. 2004) and chloroplast large subunit (23S)-rDNA (Santos et al. 2002), all of which produced congruent phylogenies. Recently, fine-scale molecular markers have revealed additional diversity within Symbiodinium. For example, LaJeunesee (2001) divided members of each clade into several different ?types? by denaturing gradient gel electrophoresis (DGGE) of ITS 2 (LaJeunesse 2001). At the population level, microsatellite loci specific to Symbiodinium Clade B have detected even finer-scale genetic differences and specificity between Symbiodinium and hosts such as Caribbean octocorals (Santos et al. 2004; Pettay & LaJeunesse 2007; Xiang et al. 2009). 5 Thus, additional genetic diversity within Symbiodinium will likely be revealed as molecular methodologies improve in the future. To date, most systematic work on Aiptasia has been based on morphological characters. Previous Aiptasia studies focused on two abundant species, A. pulchella and A. pallida, which are geographically separated. Aiptasia pulchella is reported to be distributed across the Pacific Ocean, India Ocean and Red Sea, whereas A. pallida is distributed throughout the Atlantic Ocean and Caribbean (Oskar 1943, 1952; Cutress 1955). However, current debate concerns whether these two species are synonymous.. Thus, molecular data may prove useful in identifying if and where species boundaries exist between them, which will contribute toward our understanding of specificity between Symbiodinum and Aiptasia, as well as their co-evolutions. Specificity and flexibility of the symbiosis: Specificity, member of same host taxa harbors specific symbionts only, has been reported in many host-Symbiodinium symbioses. Examples include: the scyphistoma stage of the jellyfish Cassiopeia xamachana selectively phagocytes only particular symbiotic algae (Colley & Trench 1983); the density of Symbiodinium from the same host associated with temperate sea anemone Cereus pedunculatus reaches higher densities than heterogeneous Symbiodinium (from different host) in host cells (Davy et al. 1997); the aposymbiotic planulae of the temperate anemone Anthopleura elegantissima are only capable of forming an association with fresh algal isolates from a conspecific adult (Banaszak et al. 1993; Schwarz et al. 1999); and gorgonians such as Plexaura kuna and Pseudoplexaura porosa harbor members of Symbiodinium Clade B after three 6 months although newly settled polyps naturally acquire Symbiodinium Clades A, B and C (Coffroth et al. 2001). Similarly, specificity was also described in Aiptasia spp. (Schoenberg and Trench 1980a-c). These authors found aposymbiotic A. tagetes were more successfully infected by a single taxon of Symbiodinium (from same host species) than by heterogenous isolates (from different host), while some Symbiodinium were unable to infect individual anemones at all even after six months of inoculation and exposure. Interestingly, algal isolates from identical or closely-related anemones seem to be favored in associations with the Aiptasia hosts (Belda-Baillie et al. 2002). Although specificity between symbiotic dinoflagellates and their hosts has been documented in various studies, mixed assemblages can also be established (Banaszak et al. 1993; Schwarz et al. 1999). For example: Aiptasia hosts may associate with algal isolates from tridacnid clams to a limited extent (Belda-Baillie et al. 2002). Numerous other marine invertebrates can also host Symbiodinium from mixed asemblages under specific, labortory conditions (Schoenberg & Trench 1980a-c; Colley & Trench 1983). However, over extended periods of time, these associations appear to be less stable. While limited, such flexibility in these symbioses may help hosts establish symbioses with new partners over evolutionary time. In general, there have been more studies reporting specificity than flexibility in associations between Symbiodinium and their hosts. Other examples of this specificity include reports between Foraminifera-Symbiodinium (Garcia-Cuetos et al. 2005), Madracis mirabilis-Symbiodinium (Diekmann et al. 2003) and scleractinian-Symbiodinium (LeJeunesse 2004). Future studies will likely report on the existence and extent high specificity in these associations. 7 III. ESTABLISHMENT AND MAINTENANCE OF SYMBIOTIC RELATIONSHIP Recognition between Aiptasia and Symbiodinium: Trench et al. in 1981 was the first to propose the potential recognition mechanism of symbioses between marine invertebrates and Symbiodinium (Trench et al. 1981). This mechanism was then illustrated in jellyfish Cassiopeia xamachana, which involves selective phagocytosis and persistence of particular Symbiodinium lineages (Colley & Trench 1983). Additional work in other algal symbioses has demonstrated that surface molecules on symbiont cells are crucial factors for establishment of symbiotic relationships (Meints & Pardy 1980; Reisser et al. 1982). These surface molecules and their carbohydrate groups were subsequently indicated to be involved in cell recognition (Weis & Drickamer 1996). Recent studies on the Aiptasia-Symbiodinium symbiosis have demonstrated that glycoproteins on the algal cell wall play pivotal roles in the establishment of the association (Lin et al. 2000). More detailed information on the glycoprotein-like structure, amino acid sequence and mechanism of how glycoprotein works in the recognition process are needed since their exact roles are still unknown (Lin et al. 2000; Belda-Baillie et al. 2002). 8 Maintenance of Symbiotic Relationship: Within a host cell, Symbiodinium are enclosed individually in the symbiosome, a phagosome-derived organelle. This organella provides the algal symbiont protection from herbivores and access to essential inorganic nutrients for photosynthesis. These situations likely contribute to their apparent ecological dominance over other free-living unicellular photosynthetic algae in the tropical reef ecosystem. However, how does Symbiodinium survive phagocytosis (when they get in cells of their hosts) and take up essential nutrients across the phagosome membrane for growth and replication? One possibility involves the use of particular proteins. For example, Rab (a member of the Ras superfamily of monomeric G proteins) family proteins have recently emerged as key regulators of intracellular vesicle trafficking during endocytosis and exocytosis, and several members of this family have been localized to distinct intracellular structures (Pfeffer 2001; Zerial & McBride 2001). Every step of intracellular vesicular transport is thought to be mediated by distinct sets of Rab proteins (Pfeffer 2001; Zerial & McBride 2001). For example, the Rab7 protein is a key regulator of the late endocytic pathway. It is located on late endosomes/lysosomes (Chavrier et al. 1990; Meresse et al. 1995; Vitelli et al. 1997) and regulates intracellular transport from early to late endosomes (Feng et al. 1995; Mukhopadhyay et al. 1997; Press et al. 1998) and from late endosomes to lysosomes (Meresse et al. 1995). On the other hand, Rab5 regulates the fusion between clathrin-coated vesicles and early endosomes (Bucci et al. 1992; Barbieri et al. 1994), and between early endosomes (Gorvel et al. 1991). A requirement of Rab5 activity for fusion of phagosomes with endosomes was demonstrated first by Stahl and 9 co-workers (Alvarez-Dominguez et al. 1996) and then by others (Funato et al. 1997; Roberts et al. 1999). Rab11 mediates endocytic recycling (Zerial & McBride 2001). It is mainly in charge of recycling the membrane of phagosomes to fuse with plasma membrane (Zerial & McBride 2001). Rab proteins were reported to be crutial factors during establishment of invertebrate-Symbiodinium symbioses by Chen et al. (2003, 2004, 2005). They cloned and characterized Rab proteins from sea anemone A. pulchella harboring Symbiodinium. The Aiptasia homologue of Rab5, Rab7 and Rab11 (ApRab) proteins contain all the required Rab-specific signature motifs (Chen et al. 2003, 2004, 2005). Their research results supported that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Most of the phagosomes containing live Symbiodinium did not contain detectable levels of ApRab7, while most phagosomes containing killed or photosynthesis-impaired symbionts were detected by ApRab7 staining. For ApRab5, immunofluorescence study showed that the majority of phagosomes containing live Symbiodinium were labeled with ApRab5, while those containing killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining. In all cases, ApRab11 was rarely observed in the phagosomes containing healthy Symbiodinium. Overall, these data suggest that live algal symbionts actively retain ApRab5 but exclude ApRab7 and ApRab11 from their phagosomes. By that mechanism, symbionts establish and maintain an endosymbiotic relationship with their hosts and escape destruction by the cell they reside in (Chen et al. 2003, 2004, 2005). 10 IV. METABOLIC INTERCHANGES IN SYMBIOSIS Metabolic interchanges in algal-invertebrate symbioses have been intensively studied. Generally, symbiont cells release photosynthetic carbon and may also recycle nitrogenous waste from the animal. Glycerol has been identified as a major extracellular product of Symbiodinium in numerous studies and the release of glycerol by the algal cells is thought to be stimulated by a ?host factor? that might mediate translocation in the intact association (Muscatine 1967; Muscatine et al. 1972; Trench 1979; Cook 1983). However, pathways utilized by Symbiodinium to synthesize glycerol are unknown (Trench 1979). In addition to glycerol, a variety of other photosynthetic compounds, such as Alanine and a number of other organic acids, are also released by freshly isolated Symbiodinium (Trench 1971a), with only those of low molecular weight compounds being released (Trench 1971b). Trench (1993) reported that over half of the carbon fixed by Symbiodinium is transferred to the surrounding animal tissues and this appears to give the host enough energy to survive short periods of reduced food supply. Recycling of nitrogenous waste between Symbiodinium and their host makes coral reefs to be flourishing in nutrient poor tropical oceans. Inorganic nitrogen, phosphorus and sulfur are the three most important inorganic nutrients of those recycled, and symbiotic invertebrates have the ability to assimilate dissolved inorganic nutrients from low concentrations in the environment and retain them (Muscatine 1980). Furthermore, only intact algae-invertebrate associations have the ability to uptake these nutrients since 11 no uptake is detected by aposymbionts, or hosts lacking their algal partners (Muscatine et al. 1979). Studies have suggested that under high amino acid concentrations, uptake of excess animal-derived amino acids and resultant gluconeogenesis will overload the tricarboxylic acid cycle (TCA) cycle and cause metabolic intermediates to be excreted (Gates et al. 1995). This indicates that Symbiodinium releases photosynthates because their normal metabolism is perturbed by the uptake of animal-derived amino acids (Gates et al. 1995). However, Wang and Douglas (1997) found that taurine is a chemical signal that could mediate photosynthate release through a signal cascade. This cascade then switches carbon metabolism from an endogenous fate to export. Given this finding, further studies are needed to address mechanisms for releasing photosynthates. 12 V. BLEACHING: BREAKDOWN OF SYMBIOSIS The phenomenon of ?bleaching? was first described by Glynn in 1984 among corals in the Pacific Ocean. Bleaching, which typically involving the loss of symbiotic dinoflagellates from a host, is generally deleterious to corals and ultimately the reef community (reviewed by Brown 1997). Bleaching can either be temporary or results in a nearly permanent loss of Symbiodinium, the latter of which might lead to death of the host. If the symbionts are only partially lost, the host may recover. Causation of bleaching: Bleaching can be induced via multiple means: extremes of temperature (heat shock and cold shock), low and/or high salinity, intense irradiance, prolonged darkness, heavy metals (especially copper and cadmium), pathogenic micro-organisms or a combination of these factors (reviewed by Hoegh-Guldberg 1999; Brown 2000). Among these, temperature is one of the most common reasons for bleaching. Recent large-scale bleaching events on the world?s reefs have been attributed to elevated sea water temperature resulting from global warming, often combined with increased solar radiation (Stone et al. 1999; Walther et al. 2002). Adaptive Bleaching Hypothesis: The Adaptive Bleaching Hypothesis (ABH) was first reported by Buddemeier and Fautin in 1993. This hypothesis assumed that 1) different types of Symbiodinium have 13 different responses to environmental parameters like salinity, irradiance and especially temperature; and, 2) bleached hosts might acquire Symbiodinium that vary in response to these parameters from the environment. From the time that the ABH was proposed, many studies have focused on testing these hypotheses. Kinzie et al. (2001) conducted experimental tests on assumptions underlying the ABH. They found Clade B Symbiodinium showed decreasing growth at higher temperature while a Clade C isolate showed increasing growth rate and the responses of Clade A isolates were variable. Additionally, bleached adult hosts can acquire algal symbionts with an apparently dose-dependent relationship between the concentration of Symbiodinium and the rate of establishment of the symbiosis (Kinzie et al. 2001). These results seem to lend support to the Adaptive Bleaching Hypothesis. However, many of those studies utilized culture Symbiodinium, which has been shown to be only a subset of the original population in hospite (Santos et al. 2001). Therefore, more studies are needed to address the intact symbiosis using natural populations of symbionts. 14 VI. TEMPERATE VS. TROPICAL SYMBIOSES Sea anemones with symbiotic algae are distributed in both tropical and temperate oceans. The average biomass or Symbiodinium density in temperate anemones is lower than in tropical anemones (Davy et al. 1996). In spite of pronounced seasonal variation in light and temperature in temperate regions, Symbiodinium densities remain fairly constant on spatial and temporal scales and may even increase during winter conditions for temperate anemones (Dykens & Shick 1984; Bythell et al. 1997; Dingman 1998; Squire 2000). Symbiodinium densities in the scyphozoan Cassiopea xamachana from the Florida Keys were the same in winter and summer (Verde & MaCloskey 1998). However, Symbiodinium densities in tropical anemones appear to be more influenced to seasonal differences in light than those from temperate environments (Brown et al. 1999; Fitt et al. 2000). Additional comparative studies are suggested by Muller-Parker and Davy (2001) to further assess this point. Interestingly, the maximum photosynthetic rates of temperate and tropical Symbiodinium are similar (Muller-Parker 1984; Fitt et al. 1982), but photosynthetic efficiency in temperate anemones is less than those in tropical anemones due to lower irradiance in temperate regions. Because of the large reduction of light in winter, temperate anemone hosts receive obviously reduced carbon supplies from their Symbiodinium (Davy et al. 1996). Currently, no clear trends have been detected in the symbiont transmission modes and specificity among anemones in tropical vs. temperate seas. However, past work supports the capacity for symbiont uptake and 15 persistence in anemones is species-specific (Schoenberg & Trench 1980a-c; also see review by Muller-Parker & Davy 2001). 16 VII. SUMMARY The Aiptasia -Symbiodinium symbiosis is now being proposed as a model system to investigate the endosymbiosis responsible for one of the most productive ecosystems in the world (Weis et al. 2008). More and more studies have indicated there is specificity between hosts and Symbiodinium. The molecules responsible for this specificity appear to be glycoproteins, although the exact properties of these molecules are still unknown. Rab proteins also appear to play an important role in the symbiosis, particularly in the process of endocytosis. Again, this area also needs more studies. As a first step in further understanding the Aiptasia -Symbiodinium symbiosis as a model system, this master study evaluated the specificity and flexibility of the endosymbiosis between Symbiodinium and Aiptasia. Here, I focus on using a suite of molecular markers toward elucidating the population genetics of Symbiodinium from Aiptasia spp. in a worldwide scale. 17 LITERATURE CITED Alvarez-Dominguez C, Barbieri AM, Beron W, Wandinger-Ness A, Stahl PD (1996) Phagocytosed live Listeria monocytogenes influences Rab5-regulated in vitro phagosome-endosome fusion. 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Nature Reviews Molecular Cell Biology, 2, 107?117. 29 CHAPTER 2 HOST SPECIFICITY AND REGIONAL ENDEMICITY IN SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM, DINOPHYTA) ASSOCIATED WITH SEA ANEMENOES IN THE GENUS AIPTASIA 30 I. INTRODUCTION Success of coral reef ecosystems is dependant upon mutualistic symbioses between cnidarians and endosymbiontic dinoflagellates in the genus Symbiodinium (Muscatine 1990). Symbiodinium spp. provide approximately 90% of their hosts? energetic needs through photosyntic products (Muscatine et al. 1981). Although these mutualisms are increasingly threatened globally (reviewed in Hoegh-Guldberg 1999), Cnidarians may alter their symbiotic associations in order to acclimatize to environmental change (Buddemeier & Fautin 1993). Therefore, understanding the diversity of Symbiodinium and the degree of specificity versus flexibility in host-symbiont associations is central to the investigation of these important relationships. Although Symbiodinium was once considered to be a single species (Freudenthal 1962, Taylor 1974), a variety of biochemical, physiological, molecular genetic, and ecological approaches have demonstrated that Symbiodinium is, in fact, a diverse and heterogeneous group of dinoflagellates (reviewed in Trench 1993; Baker 2003; Coffroth & Santos 2005; Stat et al. 2006). To date, eight sub-generic clades of Symbiodinium have been described and named A, B, C (Rowan & Powers 1991a), D (Carlos et al. 1999), E, F (LaJeunesse & Trench 2000; LaJeunesse 2001), G and H (Pochon et al. 2001, 2006). Additionally, each of these sub-generic clades can be further divided into numerous ?species?, populations, or strains (reviewed in Coffroth & Santos 2005). 31 The majority of Symbiodinium diversity studies have been conducted at the level of clades or cp23S/ITS ?types? (e.g., Rowan & Powers 1991a; Carlos et al. 1999; LaJeunesse & Trench 2000; LaJeunesse 2001; Pochon et al. 2001, 2006; van Oppen et al. 2001, 2005; Santos et al. 2003a). However, only a few studies have examined the population genetic structure of Symbiodinium spp. (i.e., Santos & Coffroth 2003; Santos et al. 2004; Kirk et al. 2005; Magalon et al. 2006; Carlon & Lippe 2008; Howells et al. 2009). Considering that populations are the fundamental units of evolution (Futuyma 2005), developing an understanding of the population genetic structure of Symbiodinium is essential to discussing how these symbionts and their hosts may respond to global climate change. To date, population level studies on Symbiodinium have been focusing on scleractinian, gorgonian and soft coral species. For example, while all Symbiodinium inhabiting the gorgonian Pseudopterogorgia elisabethae from various Bahamian reefs were cp23S genotype B184 (Santos et al. 2003a), microsatellite DNA data indicated significant population differentiation among these B184 Symbiodinium when compared between reefs (Santos & Coffroth 2003). In addition, microsatellite-based populations of Symbiodinium from the gorgonian Gorgonia ventalina were stable over time, regardless of temperature treatment or disease status (Kirk et al. 2005). In contrast to these studies, Magalon et al. (2006) reported two polymorphic Symbiodinium microsatellite loci that showed high levels of within host-colony diversity in the scleractinian Pocillopora meandrina. This pattern was interpreted as multiple symbiont genotypes occurring within most P. meandrina colonies. Overall these studies suggest that symbiosis between Symbiodinium and their host is generally specific at the population genetic level (with more complex situations possible in certain host species). Furthermore, in most cases, a 32 maximum of two clonal Symbiodinium populations of the same clade have been found within one host individual (Santos et al. 2004; Carlon & Lippe 2008). Although these previous studies are interesting, they are limited to local and regional geographical ranges; no Symbiodinium population genetic data is available across global scale. One potential study system to examine Symbiodinium population genetics at a global scale are sea anemones in the genus Aiptasia. Aiptasia spp. have been proposed as model organisms for studying cnidarian-dinoflagellate endosymbiosis, as these anemones occur in subtropical to tropical waters throughout the world and can be maintained and manipulated in the laboratory (e.g., Weis et al. 2008). Previous Aiptasia studies focused on two abundant species, A. pulchella and A. pallida, which are geographically separated. A. pulchella is distributed across the Pacific Ocean, India Ocean and Red Sea, whereas A. pallida is distributed throughout the Atlantic Ocean and Caribbean (Oskar 1943, 1952; Cutress 1955). Although the majority of symbiotic invertebrates acquire Symbiodinium from the surrounding environment (i.e., horizontal transmission), Aiptasia typically transmit symbionts directly from parent to offspring (i.e., vertical transmission) during an asexual reproductive process known as pedal laceration (Schwarz et al. 2002). Aiptasia spp. predominantly harbour Clade B Symbiodinium throughout the world (Santos et al. 2003a; Savage et al. 2002; LaJeunesse 2002; LaJeunesse et al. 2004), suggesting host-symbiont specificity in this symbiosis. Interestingly, symbioses involving Clades A (either alone or with B) or Clade B has also been reported in Aiptasia from the Florida Keys, indicating the possibility of symbiotic flexibility (Santos et al. 2003a; Kinzie et al. 2001; Goulet et al. 2005) in this anemone genus. 33 Because Aiptasia spp. are globally distributed, this study system is suitable for examining how Symbiodinium populations are genetically structured across a broad geographic range. Based on the results of previous molecular and population genetic studies summarized above, I hypothesized that 1) Symbiodinium associated with Aiptasia would exhibit high specificity between host and symbiont at a global scale; 2) Symbiodinium populations from different geographic locations are highly structured and localized. In order to test these hypotheses, I characterized the variation and distribution of Symbiodinium associated with Aiptasia from locations across the world using a suite of approaches: restriction fragment length polymorphisms (RFLPs) of the small subunit ribosome RNA gene (18S rDNA), denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer 2 (ITS2) region of the rDNA, flanking regions from four microsatellite loci, and allelic variation at six microsatellite loci specific for Symbiodinium Clade B. My results indicate a high degree of host-symbiont specificity and remarkable population structure throughout most of the global range of Aiptasia. 34 II. MATERIALS AND METHODS Collection of the anemone samples, Symbiodinium cultures and DNA extractions: Individual Aiptasia spp. anemones (n=356) were sampled from 18 localities in eight major geographic areas throughout the world (Fig. 2.1). Individuals were scrapped from the substrate and immediately fixed in 100% ethanol or acetone for molecular analyses. Additionally, 25 Symbiodinium Clade B isoclonal cultures (see below) were selected to serve as control DNA for the various analyses conducted in this study (culture details can be found in Table 2.1 and 2.2). Symbiodinium cultures were maintained in f/2 media (Guillard & Ryther 1962) at a constant temperature of 29 ?C, irradiance level ~80 ?M photons?m -2 s -1 , and a photoperiod of 12:12-h light-dark cycle prior to molecular analysis (Santos et al. 2001). Genomic DNA from the anemone samples, which included host and symbiont nucleic acids, as well as from Symbiodinium cultures, were extracted using 2? CTAB according to methods of Coffroth et al. (1992). 18S rDNA RFLP: For all Aiptasia samples (n=356), Symbiodinium small subunit (18S) ribosome DNA was amplified via the polymerase chain reaction (PCR) using the primers ss5 and ss3z (Table 2.3) according to the protocol of Rowan and Powers (1991b). Amplification products were verified by 1% sodium borate (SB) agarose gel electrophoresis (Brody & Kern 2004). Successful amplifications were digested for 3.0 hr with 0.12 U/?L of Taq 35 I (Fermentas Hanover MD, USA) and electrophoresed on 1% SB agarose gels to generate RFLP profiles, according to the protocol of Rowan and Powers (1991b). Symbiodinium clades were identified by comparison to restriction digests of cultured standards from Clades A, B and C. ITS2-rDNA denaturing gradient gel electrophoresis: The internal transcribed spacer 2 region (ITS 2) of nuclear ribosomal DNA was used to further discriminate molecular ?types? of Symbiodinium on a representative subset (n=16) of the DNA extracts from field collected populations (sample details provided in Table 2.4) (LaJeunesse 2001, 2002). The ITS2 region was amplified from these DNA extracts for denaturing-gradient gel electrophoresis (DGGE) using primers "ITS 2 clamp" (5' - CGCCCGCCGC GCCCCGCGCC CGTCCCGCCG CCCCCGCCCG GGATCCATAT GCTTAAGTTC AGCGGGT - 3') and "ITSintfor 2" (5' - GAATTGCAGA ACTCCGTG - 3') (LaJeunesse & Trench 2000). PCR was performed under the following conditions: 0.5?1.0 ?L DNA, 2.5 ?L 10? PCR buffer (Eppendorf), 2.0 ?L 25 mM Mg(OAC) 2 , 2.5 ?L 2 mM dNTPs, 0.25 ?L 10 ?M ?ITSintfor2?, 0.5 ?L 10 ?M ?ITS2CLAMP?, 0.15 ?L 5 U/?L Taq DNA polymerase, and distilled water to a total volume of 25 ?L per reaction. Amplification used the following protocol: initial denaturation 94 ?C, 3 min; 40 cycles of denaturation 94 ?C, 40 s; variable annealing temperature (see below for ?touchdown? conditions), 40 s; extension 72 ?C, 30 s; final extension 72 ?C, 10 min. For annealing temperatures, ?touchdown? conditions 10 ?C above the final annealing temperature of 52 ?C was used to ensure PCR specificity. The annealing temperature was then decreased by 0.5 ?C after each of the first 20 cycles. 36 Once the annealing temperature reached 52 ?C, it was maintained at that setting for another 20 cycles. All PCR amplifications were verified by 1% SB agarose gel electrophoresis prior to DGGE analysis. DGGE gels were poured (following manufactorer?s instructions) using 8% polyacrylamide (37.5:1 acrylamide/bisacrylamide ratio), approximately 20 cm long plates, 0.75 mm spacers, and a 45-80% denaturing gradient (100% denaturant contains 7 mol L -1 urea and 40% deionized formamide). Prior to loading samples on the DGGE gel, excess denaturant was purged from wells using a micropipette. 20 ?L of each PCR reaction was added to 10 ?L of xylene cyanol loading dye (pH 7.0) and a total of 10 ?l of the combined product was loaded onto each DGGE gel. Runs were performed at 60 ?C. The temperature within the buffer chamber was checked prior, during, and after the run at multiple positions to exclude biases caused by incomplete heating. Gels were electrophoresed at 150 V for 10 h (1500 Vh) on a C.B.S. Scientific? DGGE-1001 model apparatus. All DGGE gels were stained with SYBR Green (Molecular Probes, 10,000? diluted in 1? TAE) for 20 min and photographed under UV light using a digital camera fitted with a SYBR Green filter. To identify symbiont types, the DGGE fingerprint for each sample was compared to ITS2 standards from clonal Symbiodinium cultures. Sequencing of microsatellite flanking regions: The microsatellite flanking regions from five out of six loci (see below) were sequenced from the Symbiodinium of representative Aiptasia spp. samples (n=13) and cultures (n=11) (details on these cultures provided in Table 2.2; culture selection was based on Santos et al. [2004]). Santos et al. (2004) reported that the flanking region 37 sequence of locus CA6.38 was not variable between Symbiodinium from two phylogenetically divergent hosts, therefore, sequence analyses were limited to the flanking regions of the remaining five loci (i.e., CA4.86, Si4, Si8, Si15, and Si34). Details regarding the PCR primer sequences, annealing temperatures, MgCl 2 concentrations and references are presented in Table 2.3. PCR reactions for these five loci were performed in 10 ?L volumes containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.001% gelatin, 200 ?M dNTP, 0.5 U Taq polymerase, 0.60 ?M forward primer and reverse primer and 10 ng of template DNA. Thermocycling conditions were as follows: 2 min at 94 ?C for initial denaturing, 32?40 cycles of 94 ?C for 30 s, a variable annealing temperature (see Table 2.3) for 30 s and 72 ?C for 30 s, followed by 5 min at 72 ?C for final extension. Amplifications were purified using Montage? PCR Filter Units (Millipore) and DNA sequenced using Big-Dye Terminators and read on a PRISM 3100 Genetic Analyzer (Applied Biosystems). Raw sequence data were assembled using SEQUENCHER 4.7 (Gene Codes) and finished sequences were aligned automatically using CLUSTAL_X (Thompson et al. 1997) or manually using SE-AL VERSION 2.0a11 (available at http://tree.bio.ed.ac.uk/software/seal/). All sequences have been deposited in GenBank (Accession nos. XXXXXX-XXXXXX; Table 2.2. Chapter 2 will be submitted to Molecular Ecology. Note that accession nos. will be added upon acceptance the manuscript). Data from the flanking regions of several microsatellite loci were excluded from the analysis for the following reasons. First, attempts to sequence the Si4 locus from numerous Symbiodinium samples were unsuccessful. Furthermore, locus Si8 possessed no variation in any of the examined Symbiodinium cultures or Symbiodinium sample from 38 Aiptasia. Finally, all variable sites in the flanking regions of locus Si34 were four base pair indels. This suggests that flanking region variability is likely due to differences in the number of microsatellite repeats, indicative of non-phylogenetically informative allelic variation. Thus, loci Si4, Si8, and Si34 were excluded and phylogenetic analyses were conducted based on flanking region sequences from loci CA4.86 and Si15. Maximum-parsimony (MP) analyses were performed in PAUP4.0b10 (Swofford 2002), with ten additional replicates using stepwise addition to obtain starting trees and Tree-Bisection-Reconnection (TBR) to swap branches. Maximum-likelihood analyses were also performed in PAUP4.0b10 with a GTR+?+I model, as recommended by MODELTEST v3.7 based on the Akaike information criterion (AIC) (Posada & Crandall 1998). Heuristic searches were run with ten random-taxon replicates using TBR swapping. All model parameters used fixed values as recommended by MODELTEST v3.7. Branch supports in MP and ML trees were estimated by bootstrap analysis of 1000 replicates in PAUP4.0b10. Microsatellite size fragment analysis: Six Symbiodinium spp. Clade B microsatellite loci (loci CA4.86 and CA6.38 [Santos & Coffroth 2003] as well as loci Si4, Si8, Si15 and Si34 [Pettay & LaJeunesse 2007]) were used to quantify population genetic differences in 234 Aiptasia samples harbouring Clade B Symbiodinium in 17 of the 18 sampling locations (Aiptasia population WK1 from Florida only contains Symbiodinium Clade A). Furthermore, all six microsatellite loci were also screened against 16 clonal Symbiodinium cultures, each of which originated from a single cell (details on the cultures can be found in Table 2.1). 39 Cultures were selected based on Santos and Coffroth (2003) in order to provide DNA from a single genetic entity to serve as control DNA. Finally, to test for background populations of Symbiodinium Clade B in Florida Aiptasia, the six microsatellite loci were tested on 50 random selected Aiptasia individuals from Florida whose RFLP analysis implied the sole presence of Symbiodinium Clade A in that host individual. Sequences of PCR primers, annealing temperatures, and MgCl 2 concentrations used in these analyses are presented in Table 2.3. PCR reactions for these six loci were performed in 10 ?L volumes containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.001% gelatin, 200 ?M dNTP, 0.5 U Taq polymerase, 0.15 ?M WellRED D2, D3 or D4 fluorescent-labeled M-13 primer (Sigma-Proligo), 0.30 ?M forward primer, 0.15 ?M reverse primer and 10 ng of template DNA. Nineteen nucleotides (5?-CACGACGTTG TAAAACGAC-3?) were added to the 5? end of reverse primers to allow the incorporation of the M13 fluorescent-labeled primer into PCR products. In all other cases, amplification conditions were identical to those described above for microsatellite flanking regions. Microsatellite allele size determinations were performed on CEQ-8000 Genetic Analysis System (Beckman Coulter) under the default fragment analysis parameters. Each well contained 4 ?L of PCR product, 20 ?L sample loading solution (Beckman Coulter) and 0.5 ?L 400 base pairs (bp) DNA size ladder (Beckman Coulter). Alleles were scored according to their true allele size by excluding the nineteen 5?-nucleotides of the fluorescent-labled M13 primers. Genotypes were constructed for the Symbiodinium Clade B population of each Aiptasia individual using the recovered allele sizes from each of the six microsatellite 40 loci. These genotypes were tested for linkage equilibrium using the computer program GENETIC DATA ANALYSIS (Lewis & Zaykin 2001). According to the total number of alleles observed for a locus from all samples, allelic frequencies and allelic diversities were calculated separately for each population, as well as across all populations, using the program TOOL FOR POPULATION GENETIC ANALYSIS (TFPGA) V1.3 (Miller 1997). F ST and R ST , which assume infinite-alleles (IAM) and stepwise-mutation (SMM) evolutionary models, respectively, were estimated by using ? and ? ST respectively under FSTAT V2.9.3.2 (Goudet 2001). The standardization approach of Goodman (1997) was used to make a six-locus measure of R ST . Pairwise tests for Symbiodinium sp. clade B population differentiation were also conducted by randomizing genotypes between pairs of populations using FSTAT V2.9.3.2. Multiple simultaneous comparisons were corrected by using sequential Bonferroni corrections (Rice 1989). To graphically describe the relationship between Symbiodinium clade B populations in Aiptasia anemones, an unweighted pair group method using arithmetic averages (UPGMA) dendrogram was constructed using Nei?s minimum genetic distance (Nei 1972) in TFPGA. 41 III. RESULTS Symbiodinium clades associated with Aiptasia spp.: Symbiodinium 18S rDNA RFLP analysis of Aiptasia spp. from 18 locations throughout the world implies that Symbiodinium Clade B is widely associated with Aiptasia spp. around the globe, including in the Pacific Ocean (i.e., Hawai?i, Australia, Japan, and Mexico), the Indian Ocean (Thailand), the Red Sea, and the western Atlantic Ocean (Bermuda, with the exception of the Florida Keys, see below) (Table 2.4). This result suggested that the symbiosis between Symbiodinium and Aiptasia host is generally specific. More complex patterns of symbiotic associations occurred in Aiptasia from the Florida Keys (Table 2.4). In contrast to Aiptasia spp. from the rest of the world, symbiosis with Symbiodinium Clade A was most common in Florida. Furthermore, mixed symbioses of Symbiodinium Clades A and B, or more rarely Clades A and C, were also found (Table 2.4); no such mixed symbioses were observed in regions other than the Florida Keys. Symbiodinium ITS2-DGGE profiling: To further discriminate molecular ?types? of Symbiodinium within Clade B, PCR-DGGE analysis of the internal transcribed spacer 2 region (ITS2) of nuclear ribosomal RNA genes (LaJeunesse & Trench 2000, LaJeunesse 2002) was employed on a 42 subset of samples (Table 2.4). All samples examined (n=18) were detected as harbouring only ITS2 type B1 (sensu LaJeunesse 2001). Therefore, for ITS2, the symbiont assemblage did not vary among anemones hosting Symbiodinium Clade B , at least within the detection limits of DGGE (see Thornhill et al. 2006b) Symbiodinium microsatellite flanking regions: Flanking regions from the Symbiodinium Clade B microsatellite loci CA4.86 and Si15 were sequenced from samples of Aiptasia spp. throughout the world (n=13). From 359 base pairs recovered from the regions flanking these two loci, no variation (either base pair substitutions or indels) was found, regardless of region sampled. Thus, unique flanking region phylotypes of these two loci from Symbiodinium Clade B associates with Aiptasia anemones across the entire global range of this host, again indicating specificity in this symbiosis. In order to confirm that variability exists in the flanking region sequences of other ITS2 ?type? B1 Symbiodinium, 11 clonal Symbiodinium Clade B cultures (of ITS2 ?type? B1) were selected for sequencing of microsatellite flanking region loci CA4.86 and Si15 (see table 2.2). The combined dataset consisted of 359 nucleotide positions, 100% of which could be unambiguously aligned. Considerable variation was encountered between the cultured isolates. Specifically, 25 characters (7.0%) were variable, among which 24 (6.7%) were parsimony informative. Consequently, the lack of variation in flanking regions from Symbiodinium Clade B hosted by Aiptasia cannot be attributed to a lack of potential for polymorphisms in these molecular markers. 43 To infer phylogenetic relationships among the Clade B Symbiodinium of Aiptasia and the 11 cutures, an unrooted phylogenetic tree was reconstructed by using MP and ML methods (Fig. 2.2). All Symbiodinium Clade B from Aiptasia (including field samples and culture FLAp2, the latter of which was isolated from Florida Aiptasia) clustered into a single group, with no nucleotide variation between samples. In contrast, the 11 Symbiodinium cultured were distributed among six groups. Interestingly, Symbiodinium from Aiptasia were most closely related to Symbiodinium cultured from the gorgonians Plexaura kuna, Gorgonia ventalina and Pseudoplexaura porosa. Population genetic structure of Symbiodinium associated with Aiptasia spp.: Analyses of six microsatellite loci (i.e., CA4.86, CA6.38, Si4, Si8, Si15, and Si34) from 16 clonal Symbiodinium cultures detected only a single allele from each clonal culture (Table 2.1). This is consistent with previous work that reported Symbiodinium spp. are haploid (Santos & Coffroth 2003). Therefore, based on results from clonal cultures, it is reasonable to infer that if a single clonal line is harboured by an Aiptasia individual, only one allele would be identified per Symbiodinium Clade B population in hospite. The six microsatellite loci were examined from a total of 234 Aiptasia individuals harbouring Symbiodinium Clade B (Table 2.5, detailed results see appendix table). For most Aiptasia samples, a single allele was recovered from each Symbiodinium Clade B populations (n=180, 76.9% of the colonies). While significant linkage disequilibrium was found in population CK2 (CA6.38/Si15, CA6.38/Si34, Si15/Si34) from the Florida Keys and population RS (CA6.38/Si8, CA6.38/Si15, Si8/Si15) from the Red Sea, this pattern was not found in these loci across all other sampling locations. Thus, I conclude that 44 these loci are sorting independent of each other. Although locus CA4.86 was found to be monomorphic, regardless of the population sampled, allelic variation was identified in the remaining five loci (Table 2.5). For instance, four different alleles occurred at locus CA6.38, with the frequency of alleles varying significantly by region. The most polymorphic locus was Si34, which included six different alleles. Allele diversity, estimated by heterozygosity (H), for each locus ranged from 0 to 0.5. The average H for the six loci across all populations was 0.410, indicating high levels of genetic variation in Symbiodinium Clade B associated with Aiptasia spp. (Table 2.6). Strong subdivision in the Symbiodinium Clade B populations of Aiptasia spp. was indicated from estimates of population structure estimating by F ST and ? ST values (Table 2.7). Symbiodinium Clade B populations associated with Aiptasia differed significantly from one another across their global range (Table 2.5). A total of 32 unique genotypes were identified from the 17 geographic localities. Populations from Hawai?i, Florida, Bermuda, and the Red Sea were comprised of multiple genotypes, whereas populations from Mexico, Japan, Thailand, and Australia possessed a single genotype. In most cases, Aiptasia harboured site-specific Symbiodinium genotypes, indicating that these Symbiodinium populations are regionally structured. For example, all Symbiodinium genotypes in Aiptasia populations from Australia, Bermuda, and Thailand were unique when compared to all other localities. Most populations from the Florida Keys, Red Sea, and Hawai?i were also unique compared to other localities. Similar results were inferred from the statistical pairwise test (Table 2.8). Of the 136 potential pairwise combinations, 77 were significant (71.3%). Among non-significant comparisons, 97.9% involved populations from Hawai?i, Mexico and Florida. The 45 remaining non-significant comparisons were between populations in the Red Sea (i.e., RS and EA). In general, results from the pairwise comparisons also supported significant population genetic structure occurred among Symbiodinium Clade B populations in Aiptasia. Furthermore, the UPGMA dendrogram identified sites sharing the same genotype as grouping together, while geographically proximate sites (i.e., populations RS and EA from Red Sea, populations CK and WK from Florida) also clustered closely together (Fig. 2.3). Interestingly, the Symbiodinium Clade B populations from Aiptasia collected at CI1 and WA in Hawai?i as well as at sites in western Mexico and all populations from Japan shared the same genotype (Table 2.5, Fig. 2.3). Along with this, one algal genotype was shared between population CK2 in Florida and population RS in the Red Sea. Thus, while little gene flow at the population level was observed between most geographic regions, some genotypes had wide distributions. Occurance of mixed Symbiodinium populations: In some cases (n=54 out of 234, 23.1% of hosts), two alleles were recovered in four out of the six microsatellite loci (including CA6.38, Si8, Si15, and Si34). Because Symbiodinium is likely haploid (Santos & Coffroth 2003), multiple alleles are indicative of mixed symbiont populations. Regionally, multiple alleles occurred in six of the seventeen sampling localities. For instance, most of the Aiptasia from Coconut Island in Hawai?i (21 of 23 from population CI1; 15 of 21 from population CI2) harboured various combinations of two alleles. Two alleles at locus Si34 was also recovered from other Aiptasia populations in Hawai?i (e.g., WA). In other localities, the recovery of two alleles 46 occurred in two of 24 individuals in population CK2 from Florida at loci CA6.38 and Si15, two of 14 individuals in BE population from Bermuda, and seven of 18 individuals in population RS from the Red Sea at loci CA6.38, Si8 and Si15 (Table 2.5). In all cases, a maximum of only two alleles per microsatellite locus was found in a single host individual. All six microsatellite loci were also tested on 50 random selected Aiptasia individuals from Florida whose RFLP analysis indicated the presence of only Symbiodinium Clade A. From these, eighteen out of 50 samples (36%) (i.e., 0 of 9 in population WK1, 3 of 13 in population WK2, 6 of 12 in population CK1, and 9 of 10 in population CK2) were detected as also harbouring Symbiodinium Clade B (see appendix table). This pattern suggests a frequent incidence of low density or ?cryptic? Symbiodinium Clade B (i.e., occurring at a density below the detection limits of 18S rDNA RFLP) in Aiptasia anemones from the Florida Keys. 47 IV. DISCUSSION Herein, I examined the genetic diversity of Symbiodinium from 18 localities across the global range of Aiptasia spp. Results from RFLP analysis of 18S rDNA, PCR-DGGE profiling of the ITS2 rDNA, and sequencing of two microsatellite loci flanking regions all indicated a high degree of specificity between Symbiodinium and Aiptasia spp. Furthermore, microsatellite genotypes from Symbiodinium Clade B populations demonstrated that most are regionally specific, with little gene flow between sites or regions. The specificity and regional endemicity found in this study are of particular interest because Aiptasia spp. are widely distributed hosts which have been recommended as a model system to study invertebrate-dinoflagellate symbioses (e.g., Weis et al. 2008). Specificity between Symbiodinium and Aiptasia spp.: The ability to host multiple and differing genetic and physiological lineages of symbionts, known as symbiotic flexibility, has been hypothesized to be an important adaptation in many symbiotic cnidarians (Buddemeier & Fautin 1993). It is thought that reef-building corals, anemones, and other symbiotic cnidarians that can host several different Symbiodinium spp. may be able to acclimatize to changing environmental conditions through a change in their complement of symbionts. Thus, symbiotic change should be particularly pronounced during stress events that result in a decrease in 48 Symbiodinium density, pigment concentration, or both (a phenomenon known as bleaching; Brown 1987, 1997; Glynn 1991, 1996; Buddemeier & Fautin 1993; Brown et al. 1996; Fitt et al. 2001). As a result, the degree of flexibility versus stability in the cnidarian-dinoflagellate symbiotic relationship has significant implications for the future success of coral reef environments, particularly in the context of global climate change that has contributed to increasing the frequency and severity of bleaching (Buddemeier & Fautin 1993; Glynn 1991, 1996; Brown et al. 1996). In the present study, results based on RFLP of the 18S rDNA gene, DGGE of the ITS2 region, and sequencing of the flanking regions surrounding microsatellite loci indicate a high degree of specificity between a single phylotype of Symbiodinium Clade B and Aiptasia spp. throughout the world. In fact, no other Symbiodinium clades or phylotypes were found at any of the depths, habitats, or regions sampled, with the sole exception of the Florida Keys. Thus, this specific relationship suggests that when stress events occur, most Aiptasia spp. will not be able to acquire alternative Symbiodinium phylotypes through either exogenous ?switching? or endogenous ?shuffling? of symbiont species (Baker 2003). Symbiotic stability and specificity has been reported in a number of previous Symbiodinium diversity studies (e.g., Goulet & Coffroth 2003; Santos et al. 2004; Thornhill et al. 2006a,b). Indeed, a meta-analysis of available Symbiodinium diversity data suggests that most hosts (~75% of host species) only harbour a single symbiont clade throughout their entire range (Goulet 2006, 2007; but see Baker & Romanski 2007). Data from this study suggest that most Aiptasia spp. also harbour a single clade or phylotype of symbiont, again with the notable exception of hosts from the Florida Keys. 49 Global biogeography of Clade B Symbiodinium populations associated with Aiptasia: Identical cp23S-rDNA and ITS2-rDNA ?types? have been recovered from Indo-Pacific and Atlantic oceanographic basins (e.g., LaJeunesse 2001, 2002, 2005; Santos et al. 2001, 2002; LaJeunesse et al. 2003, 2004), suggesting widespread dispersal of certain Symbiodinium spp. over evolutionary time. My results, based on 18S-rDNA RFLP, ITS2-rDNA DGGE, and microsatellite flanking region sequences, also support long-term dispersal capacity of certain Symbiodinium lineages, in this case a specific sub-type of ITS2 ?type? B1 (sensu LaJeunesse 2001). Despite this, data from six microsatellite loci indicate that Aiptasia spp. in most localities harboured regionally endemic Symbiodinium Clade B genotypes and populations. Strong population structure was observed across local (e.g., ~30 km between populations CK1 and CK2 in Florida), regional (e.g., ~1,800 km between populations in Florida versus Bermuda), and global (e.g., >14,000 km between Florida and Okinawa, Japan) geographic scales. Here, significant population structure detected across local to global geographic scales indicates that Symbiodinium associated with Aiptasia likely have low dispersal capacities over evolutionary time scales (Santos et al. 2003b). If this is the case, a lack of symbiont dispersal capacity could pose a considerable impediment to the future success of coral reef ecosystems. Specifically, symbiotic cnidarians likely experience significant Symbiodinium population bottlenecks following bleaching events due to the expulsion or death of symbionts. If Symbiodinium populations are unable to disperse widely over ecological time scales to replace those lost due to bleaching, the resulting loss of genetic diversity may further exacerbate the damage bleaching events cause to coral reef communities. Therefore, Symbiodinium population connectivity should be investigated in 50 other symbiotic hosts, particularly reef-building corals, to better determine the dispersal and recovery capacity following severe bleaching events. In contrast to the genetic structure observed across most regions, Aiptasia populations from some sites in Hawai?i, Mexico, and Japan shared the same Symbiodinium genotype. One potential reason for this shared genotype is oceanic currents driving gene flow between widely separated Pacific locations. Previous studies also proposed that currents affect the population structure of Symbiodinium across spatial scales. For instance, Santos et al. (2003b) suggested that the similarity of Symbiodinium populations in the gorgonian P. elisabethae among Bahamas islands was due to currents and tidal flow. Magalon et al. (2006) found a significant correlation between the Pocillopora meandrina symbiont F ST values and distance matrices, suggesting ocean currents are likely driving population structure in Symbiodinium from the Society Archipelago. In the current study, Aiptasia populations from Hawai?i, Mexico and Japan may experience gene flow among these Pacific localities. However, genotype sharing did not occur in most of the sampling locations. Given the large geographic distances and potential physical oceanographic barriers (i.e., the East Pacific Barrier; Ekman 1953) between Hawai?i, Mexico and Japan, the possibility of gene flow among those localities may seem unlikely. Alternatively, the observed connectivity may be due to transport via ballast water or some other anthropogenic mechanism rather than natural dispersal. These two hypothesis could possibly also explain the shared genotype between population CK2 in Florida and population RS in the Red Sea. 51 Mixed symbiont populations within host individuals: Occurrence of multiple Symbiodinium ?types? within host individuals has been detected in a number of host species (Goulet 2006 and references within). In previous investigations of Aiptasia, up to two different Symbiodinium clades (i.e., Clades A and B or Clades A and C) were detected to be simultaneously associated with Aiptasia from Florida (e.g., Santos et al. 2001, 2003a; Goulet et al. 2005). These previous findings are also supported by data from the present study for Aiptasia spp. in Florida. However, Aiptasia from other regions of the world appeared to be considerably less flexible in their symbioses. One reason for this might be that host mutations and subsequent selective forces on Aiptasia in Florida have produced a host species more capable of symbiont change (whether ?switching? or ?shuffling?) than those from elsewhere. Symbiotic flexibility in Florida Aiptasia may be physiologically advantageous to the host, particularly in response to changing environmental conditions, by enabling changes in the community of symbiotic dinoflagellates (see Buddemeier & Fautin 1993). For Aiptasia spp. from other regions of the world, the stable symbiosis observed here may make predictions of performance under stressful conditions less optimistic. However, detailed studies of the performance of various host-symbiont combinations under stressful temperature and light conditions are necessary to validate this conjecture. Another question that arises from the biogeographic patterns of symbiotic associations in Aiptasia spp. is why symbioses in animals from Florida are so different with those from all other locations. One explanation is that Aiptasia from Florida are genetically distinct relatively to those from the rest of the world. Since Aiptasia?s original description (Gosse 1858), the taxonomy of this genus has undergone considerable 52 revision (see Daly et al. 2003). While the current paradigm of an Atlantic species (A. pallida) and an Indo-Pacific species (A. puchella) has dominated the recent literature, no molecular genetic studies have been conducted to validate this situation. Consequently, future studies focused on the molecular genetics of the host anemones are warranted. I have preliminarily investigated this topic using inter simple sequence repeats (ISSR) in an attempt to determine the genetic structure of Aiptasia from across the world (see conclusions). Most studies on mixed Symbiodinium communities within a host have been conducted using molecular genetic markers that measure Symbiodinium diversity at the level of sub-generic clade, phylotype, or approximate ?species? (e.g., Coffroth et al. 2001; van Oppen et al. 2001, 2005; LaJeunesse 2002; Goulet 2006; Thornhill et al. 2006a,b; Meiog et al. 2007). Few studies have focused on mixed symbiosis using finer-scale population level markers. In my study, only a single ITS2/flanking region phylotype of Symbiodinium Clade B was found associated with most Aiptasia spp., indicating specificity at higher levels. Despite this, up to two Symbiodinium Clade B microsatellite genotypes, indicating two different clonal populations, were found to coexist in a single Aiptasia individual. In one instance, a single Aiptasia anemone from Florida associated with two different Symbiodinium Clade B genotypes as well as (at least) one member of Symbiodinium Clade A. Therefore, up to three distinct symbiont populations (two Clade B populations and one or more Clade A populations) were found to simultaneously occur within an individual Aiptasia host. This maximum of two populations from a single sub-generic clade being found within an individual host is a finding supported by several previous studies (i.e., Santos et al. 2004; Carlon & Lippe 2008; but see Magalon et al. 53 2006). Using Symbiodinium microsatellite flanking regions, Santos et al. (2004) found that different symbiont ?types? were each specific to different Caribbean octocoral species, with a maximum of two clonal symbiont lineages detected within a single host colony. Similar results were also found in the stony coral Favia fragum, where no more than two populations of Clade B Symbiodinium were detected within a single colony (Carlon & Lippe 2008). This apparent limit of two populations per host may be driven by competition interactions between symbionts, where slower growing strains are displaced by competitive dominants (e.g., Fitt 1985). Cryptic level background Symbiodinium Clade B in Aiptasia from Florida: Low levels Symbiodinium Clade B were detected in 18 out of 50 Florida Aiptasia individuals whose RFLP profile indicated only the presence of Clade A. These ?cryptic? or background populations of symbionts at low density in Aiptasia are analogous to a recent study that reported background levels of Symbiodinium Clade D in several symbiotic corals (Mieog et al. 2007). Given the highly structured populations in Symbiodinium associated with Aiptasia, these ?cryptic? symbionts may represent alternative symbiotic combinations via ?shuffling? (sensu Baker 2003). However, the presence of low-density symbionts alone provides no indication of the role these symbionts play physiologically in Aiptasia. Further study of these background Symbiodinium populations is necessary in order to elucidate the significance in physiological contributions to the host. 54 V. CONCLUSIONS Here, it was hypothesized that symbioses between Aiptasia and Symbiodinium would be highly specific and exhibit strong population structure between different geographic locations. My results were generally consistent with these hypotheses. With the exception of Florida, Aiptasia spp. associated with a single phylotype of Symbiodinium throughout the global range of this host. Analyses of six microsatellite loci further suggested that Symbiodinium populations are highly endemic, with the exception of potential population connectivity between sites across the Pacific Ocean (Japan, Hawai?i, and Mexico). The population genetic structure, lack of gene flow, and symbiotic stability observed here has important implications. 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Molecular Ecology, 14, 2403?2417. van Oppen MJH, Palstra FP, Piquet AMT, Miller DJ (2001) Patterns of coral-dinoflagellate associations in Acropora: significance of local availability and physiology of Symbiodinium strains and host-symbiont selectivity. Proceedings of the Royal Society of London Series B-Biological Sciences, 268, 1759?1767. 64 Weis VM, Davy SK, Hoegh-Guldberg O, Rodriguez-Lanetty M, Pringle JR (2008) Cell Biology in Model Systems as the Key to Understanding Corals. Trends in Ecology and Evolution, 23, 369?376. 65 SUMMARY Marine invertebrates and their symbiotic dinoflagellates in the genus Symbiodinium have been intensively studied in recent years. However, the degree of specificity and flexibility between partners remains unclear. In this master thesis, a comprehensive review of the symbiosis between Symbiodinium and Aiptasia was conducted. This work focused on the revealing of population genetics of the symbionts. Specifically, restriction fragment length polymorphism (RFLP) analyses were first utilized to quantify the diversity in symbiont populations from 356 Aiptasia individules that were collected from 18 localities worldwide. Aiptasia from the Florida Keys were found to host either Symbiodinium Clades A, B or mixtures of both A and B simultaneously while Aiptasia from all other locations harbored Clade B only. To quantify fine-scale population structure and genetic differences among the symbiont populations, six microsatellite loci specific for Symbiodinium Clade B were utilized on 326 individual Aiptasia. Strong population structure in Clade B populations was observed since most genotypes were unique to a specific locality. However, no sequence variation was observed in the flanking regions of these loci, suggesting an identical Symbiodinium Clade B phylotype associates with Aiptasia on a worldwide scale, which implies high specificity in this invertebrate-algal symbiosis. Additionally, I found that 18 out of 50 (36%) Florida Aiptasia thought to harbor only Clade A by RFLP analyses also possessed low levels of Clade B symbionts when examined by microsatellite analyses, suggesting 66 background symbiont populations of a host may escape detection depending on the utilized technique. Coinciding with the distinction of Symbiodinium between Florida and all other locations, preliminary data (using inter simple sequence repeats, ISSR, techniques on nuclear sequences) focusing on the population genetics of the host, Aiptasia spp., suggest that Florida Aiptasia are genetically distinct from all other localities, implying a high specificity of the symbiosis between Symbiodinium and Aiptasia. Additionally, the genectic difference of Aiptasia from Florida and other localities indicates that this genus is comprised of two ?genetic? species. Notably, the distribution of the ?genetic? species does not coincide with the range of the morphologically described species A. pulchella (Pacific and Indian Oceans and Red Sea) and A. pallida (Atlantic Ocean and Caribbean Sea). For this reason, further studies are needed using additional molecular markers to investigate the population structure of the host, Aiptasia, which will be important in better understanding the specificity and flexibility of this cnidrian-Symbiodinium endosymbiosis. Generating better ISSR markers for quantifying population structure seems time-consuming since optimizing ISSR reaction conditions, cloning and sequencing target fragments, as well as selecting appropriate fragments are all not easily to be done. Compared with ISSR, microsatellite markers may provide more informative population structures although generating microsatellite markers needs considerable efforts as well. 67 TABLES 68 Table 2.1. Information on Symbiodinium cultures used in analyses of the six microsatellites specific to Clade B. The host from which the culture was isolated, location of isolation, and microsatellite analysis results are included. Culture name Host organism Location CA4.86 CA6.38 Si4 Si8 Si15 Si34 Ap01 Aiptasia pulchella Okinawa 179 98 129 198 254 252 Zp Zoanthus pacificus Hawai?i 179 98 129 200 258 252 FLAp2 A. pallida Florida keys 179 100 131 198 258 276 FLAp2 10AB A. pallida Florida keys 179 100 131 198 258 276 208 a Plexaura kuna San Blas Islands, Panama 183 98 131 198 254 244 226 a P. Kuna San Blas Islands, Panama 183 98 131 198 254 244 595 a Briareum asbestinum Florida keys 191 102 129 200 254 256 1246 a B. asbestinum Florida keys 191 102 129 200 254 256 707 a P. Kuna San Blas Islands, Panama 191 104 129 200 254 256 1509 a B. asbestinum Florida keys 191 104 129 200 254 256 2053 a B. asbestinum Florida keys 191 104 129 200 254 256 801 P. Kuna Florida keys 193 98 129 200 246 256 13 P. Kuna Florida keys 193 98 129 200 246 256 206 a P. Kuna San Blas Islands, Panama 193 102 129 200 258 260 705 a P. Kuna San Blas Islands, Panama 193 102 129 200 258 260 SSPe Pseudopterogorgia elisabethae Bahamas 193 112 139 224 232 244 a Cultures that were from a single dinoflagellate cell. 69 Table 2.2. GenBank Accession numbers for the flanking regions of two microsatellite loci in Symbiodinium populations from Aiptasia spp. and algal cultures. GenBank Accession number Geographic locations Host name Collection sites/Populations CA4.86 Si15 BER6 a Aiptasia spp. Walsingham Pond, Bermuda XXXXXX XXXXXX CI2.2 a Aiptasia spp. Coconut Island, Hawai?i XXXXXX XXXXXX CI2.3 a Aiptasia spp. Coconut Island, Hawai?i XXXXXX XXXXXX CK1.8 a Aiptasia spp. Crawl Key, Florida XXXXXX XXXXXX CK2.3 a Aiptasia spp. Crawl Key, Florida XXXXXX XXXXXX Eilat Ap 1 a Aiptasia spp. Red Sea XXXXXX XXXXXX HERAUS1 a Aiptasia spp. Australia XXXXXX XXXXXX MXLP1 a Aiptasia spp. La Paz, Mexico XXXXXX XXXXXX RSAN1 a Aiptasia spp. Red Sea XXXXXX XXXXXX Seso Ap Lite 1 a Aiptasia spp. Sesoko Island, Japan XXXXXX XXXXXX TLAp1 a Aiptasia spp. Thailand XXXXXX XXXXXX Wsk2.4 a Aiptasia spp. West Summerland Key, Florida XXXXXX XXXXXX FLAp2 Aiptasia spp. Long Key, Florida XXXXXX XXXXXX Pk704SymB4 Plexaura kuna San Blas Islands, Panama XXXXXX XXXXXX Gv5.6a Gorgonia ventalina San Blas Islands, Panama XXXXXX XXXXXX Pp304a Pseudoplexaura porosa XXXX XXXX SSPe Pseudopterogorgia elisabethae San Salvador, Bahamas XXXXXX XXXXXX Ba06-146 Briareum asbestinum? Florida Keys XXXXXX XXXXXX Ba06-147 Briareum asbestinum? Florida Keys XXXXXX XXXXXX 04-202 ? ? XXXXXX XXXXXX 04-258 ? ? XXXXXX XXXXXX Mf10.14b.2 Montastrea faveolata ? ? XXXXXX Mf11.5b.1 Montastrea faveolata ? ? XXXXXX a Symbiodinium samples are not cultures. 70 Table 2.3. Sequence information, annealing temperatures and MgCl 2 concentrations of Symbiodinium Clade B microsatellite primers used in this study. Primer Primer sequence (5? to 3?) Study Forward (ss5): GGTTGATCCTGCCAGTAGTCATATGCTTG 18S rDNA Reverse(ss3Z): AGCACTGCGTCAGTCCGAATAATTCACCGG Rowan and Powers (1991) Forward: GCCTTCAATGCAATCACCTT CA4.86 a Reverse: GGAATTGGCCATCCCTCTAT Santos and Coffroth (2003) Forward: CAAAGAATATTCGGGGGTCA CA6.38 b Reverse: AGTTGATACGCCGGATGTGT Santos and Coffroth (2003) Forward: TCGCGATCGAGTCCCATGGTCT Si4 c Reverse: TGGTTTCCCGTGACATCCCTG Pettay and LaJeunesse (2007) Forward: ACTACAGGCACGACCCACCA Si8 c Reverse: GCATTCACGCCATCCATCAGTCC Pettay and LaJeunesse (2007) Forward: CTCACCTTGAAATCAGTAGCCA Si15 c Reverse: CGTAGCTTCTGAAGGTACGACAC Pettay and LaJeunesse (2007) Forward: TGAATGCAGTGAACGCATGG Si34 c Reverse: ACCTAGTCACCGAAGCACTC Pettay and LaJeunesse (2007) a 2.5 mM MgCl 2, 40 thermal cycles with 50 ?C annealing temperatures. b 1.5 mM MgCl 2, 40 thermal cycles with 56 ?C annealing temperatures. c 2.5 mM MgCl 2, 32 thermal cycles with 57 ?C annealing temperatures 71 Table 2.4. Clades (based on 18S-rDNA RFLP) and ITS2 ?types? (based on ITS2 DGGE) of Symbiodinium associated with Aiptasia spp. anemones from throughout the world. 18S RFLP profile ITS2 DGGE profileGeographic locations Collection sites/Populations n A B A+B A+C n ITS2 ?type? Japan Sesoko Island Dark (SD) 8 ? 8 ? ? 0 ? Sesoko Island Light (SL) 7 ? 7 ? ? 1 B1 Ishi Ap (IA) 10 ? 10 ? ? 0 ? Motobu (MA) 8 ? 8 ? ? 0 ? Tadashi Maruyama (TM) 18 ? 18 ? ? 0 ? Mexico La Paz (MX) 7 ? 7 ? ? 1 B1 Hawai?i Coconut Island (CI) 1 34 ? 34 ? ? 1 B1 Coconut Island (CI) 2 21 ? 21 ? ? 1 B1 Waikiki Aquarium (WA) 34 ? 34 ? ? 0 ? Florida West Summerland Key (WK) 1 16 16 ? ? ? 1 B1 West Summerland Key (WK) 2 44 39 ? 4 1 0 ? Crawl Key (CK) 1 33 20 4 9 ? 1 B1 Crawl Key (CK) 2 37 13 3 21 ? 1 B1 Bermuda Walsingham Pond (BE) 17 ? 17 ? ? 6 B1 Red Sea RSAN (RS) 18 ? 18 ? ? 1 B1 Eilat Ap (EA) 10 ? 10 ? ? 0 ? Thailand Thailand (TL) 8 ? 8 ? ? 1 B1 Australia Australia (HS) 26 ? 26 ? ? 1 B1 Total 18 sites 356 88 233 34 1 16 16 Table 2.5. Genotypic frequencies of six microsatellite loci in Symbiodinium Clade B associated with Aiptasia spp. from throughout the world. Genotype Site Japan Mexico Hawai?i Florida Bermuda Red Sea Thailand Australia CA4.86 CA6.38 Si4 Si8 Si15 Si34 SD SL IA MA TM MX CI1 CI2 WA WK2 CK1 CK2 BE RS EA TL HS 179 98 129 198 254 253 1.000 1.000 1.000 1.000 1.000 1.000 0.087 ? 0.563 ? ? ? ? ? ? ? ? 179 98 129 198 258 253/257 ? ? ? ? ? ? 0.130 0.048 ? ? ? ? ? ? ? ? ? 179 98 129 198/200 258 253/257 ? ? ? ? ? ? 0.043 0.095 ? ? ? ? ? ? ? ? ? 179 98/100 129 198 258 253/257 ? ? ? ? ? ? 0.043 ? ? ? ? ? ? ? ? ? ? 179 98/100 129 198 254/258 253/257 ? ? ? ? ? ? 0.174 0.048 0.375 ? ? ? ? ? ? ? ? 179 98/100 129 198/200 254/258 253/257 ? ? ? ? ? ? 0.043 0.048 0.063 ? ? ? ? ? ? ? ? 179 100 129 198 258 253/257 ? ? ? ? ? ? 0.435 ? ? ? ? ? ? ? ? ? ? 179 100 129 198/200 258 253/257 ? ? ? ? ? ? 0.043 0.048 ? ? ? ? ? ? ? ? ? 179 98 129 198 254 253/257 ? ? ? ? ? ? ? 0.048 ? ? ? ? ? ? ? ? ? 179 98 129 198 254/258 253 ? ? ? ? ? ? ? 0.048 ? ? ? ? ? ? ? ? ? 179 98 129 198 254/258 253/257 ? ? ? ? ? ? ? 0.048 ? ? ? ? ? ? ? ? ? 179 98 129 198/200 258 253 ? ? ? ? ? ? ? 0.048 ? ? ? ? ? ? ? ? ? 179 98 129 198 258 253 ? ? ? ? ? ? ? 0.238 ? ? ? ? ? ? ? ? ? 179 98/100 129 198 258 253 ? ? ? ? ? ? ? 0.095 ? ? ? ? ? ? ? ? ? 179 98/100 129 198 258 257 ? ? ? ? ? ? ? 0.048 ? ? ? ? ? ? ? ? ? 179 98/100 129 198 254/258 253 ? ? ? ? ? ? ? 0.095 ? ? ? ? ? ? ? ? ? 179 100 129 198 258 253 ? ? ? ? ? ? ? 0.048 ? ? ? ? ? ? ? ? ? 179 100 131 198 256 277 ? ? ? ? ? ? ? ? ? 0.250 ? ? ? ? ? ? ? 179 100 131 198 258 277 ? ? ? ? ? ? ? ? ? 0.250 ? 0.125 ? ? ? ? ? 179 102 131 198 256 269 ? ? ? ? ? ? ? ? ? 0.500 ? ? ? ? ? ? ? 179 102 131 198 256 273 ? ? ? ? ? ? ? ? ? ? 1.000 0.708 ? ? ? ? ? 179 100/102 131 198 256/258 273 ? ? ? ? ? ? ? ? ? ? ? 0.083 ? ? ? ? ? 179 100 129 200 256 257 ? ? ? ? ? ? ? ? ? ? ? 0.042 ? 0.111 ? ? ? 179 100 131 198 258 273 ? ? ? ? ? ? ? ? ? ? ? 0.042 ? ? ? ? ? 179 104 129 204 256/258 265 ? ? ? ? ? ? ? ? ? ? ? ? 0.071 ? ? ? ? 179 100/104 129 202/204 254/256 265 ? ? ? ? ? ? ? ? ? ? ? ? 0.071 ? ? ? ? 179 100 129 202 254 265 ? ? ? ? ? ? ? ? ? ? ? ? 0.786 ? ? ? ? 179 104 129 204 256 265 ? ? ? ? ? ? ? ? ? ? ? ? 0.071 ? ? ? ? 179 98 129 202 254 257 ? ? ? ? ? ? ? ? ? ? ? ? ? 0.500 1.000? ? 179 98/100 129 200/202 254/256 257 ? ? ? ? ? ? ? ? ? ? ? ? ? 0.389 ? ? ? 179 100 129 202 254 257 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 1.000 ? 179 102 131 198 254 253 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 1.000 n 8 7 10 8 18 7 23 21 16 4 12 24 14 18 10 8 26 72 73 Table 2.6. Heterozygosity for six microsatellite loci in Symbiodinium Clade B from Aiptasia spp. across the world. populations Heterozygosity (H) CA4.86 CA6.38 Si4 Si8 Si15 Si34 CI1 0.000 0.000 0.000 0.000 0.000 0.000 CI2 0.000 0.278 0.000 0.000 0.000 0.000 WA 0.000 0.000 0.000 0.000 0.000 0.000 MX 0.000 0.000 0.000 0.000 0.000 0.000 WK2 0.000 0.500 0.000 0.000 0.375 0.500 CK1 0.000 0.000 0.000 0.000 0.000 0.000 CK2 0.000 0.351 0.000 0.000 0.298 0.310 BE 0.000 0.153 0.000 0.153 0.153 0.000 RS 0.000 0.298 0.000 0.298 0.298 0.000 EA 0.000 0.000 0.000 0.000 0.000 0.000 TL 0.000 0.000 0.000 0.000 0.000 0.000 HS 0.000 0.000 0.000 0.000 0.000 0.000 SD 0.000 0.000 0.000 0.000 0.000 0.000 SL 0.000 0.000 0.000 0.000 0.000 0.000 IA 0.000 0.000 0.000 0.000 0.000 0.000 MA 0.000 0.000 0.000 0.000 0.000 0.000 TM 0.000 0.000 0.000 0.000 0.000 0.000 Over all populations 0.000 0.607 0.458 0.359 0.412 0.624 Table 2.7. F ST and ST ? (population differentiation) estimates of Symbiodinium Clade B from Aiptasia spp. across the global range based on six microsatellite loci. F ST ST ? CA4.86 NA a NA a CA6.38 0.844 0.924 Si4 1.000 1.000 Si8 0.919 0.979 Si15 0.816 0.890 Si34 0.920 0.965 Total 0.899 0.9517 a locus CA4.86 is not polymorphic. 74 75 Table 2.8. Symbiodinium Clade B pairwise tests of symbiont population differentiation for Aiptasia spp. at 17 sites containing Symbiodinium Clade B in the world (site abbreviations, see table 2.1; NS not significant; NA not available; * P<0.05). CI1 CI2 WA MX WK2 CK1 CK2 BE RS EA TL HS SD SL IA MA TM CI1 NS NA NA NS NS NS NS NS NS NS NS NA NA NA NA NA CI2 * * NS * * NS NS * NS * NS * * NS * WA NA NS * * * * * * * NA NA NA NA NA MX NS * * * * * * * NA NA NA NA NA WK2 * NS * * NS NS NS NS NS NS NS * CK1 NS * * * * * * * * * * CK2 * * * * * * * * * * BE * * * * * * * * * RS NS NS * * NS * * * EA * * * * * NS * TL * NS * * * * HS * * * * * SD NA NA NA NA SL NA NA NA IA NA NA MA NA TM 76 FIGURES Figure 2.1. Locations of the Aiptasia spp. populations collected from eight major geographic localities across the global range of this host. Geographic localities denoted by two-letter abbreviations as follows: HI = Hawai?ian islands; MX = Mexico; FL = Florida Keys; BR = Bermuda; RS = Red Sea; TH = Thailand; JP = Japan and AU = Australia. 77 Figure 2.2. Inferred unrooted phylogenetic relationships between Symbiodinium Clade B based on concatenated flanking regions of microsatellite loci CA4.86 and Si15. Maximum likelihood (ML) tree (?ln L = 622.68). Numbers before and after slashes are support values based on 1000 bootstrap replications (Parsimony/Likelihood respectively). For locations of Aiptasia spp. and cultures, original host name and sample locations of cultures see table 2. 78 Figure 2.3. Dendrogram by unweighted pair group method using arithmetic averages (UPGMA) depicting relationships between Symbiodinium Clade B populations of Aiptasia spp. at 17 geographic localities across the global range of the host. 79 80 APPENDIX TABLE Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 Wsk2.4 Florida A+B 179 102 131 198 256 269 Wsk2.7 Florida A+B 179 100 131 198 256 277 Wsk2.22 Florida A+B 179 102 131 198 256 269 Wsk2.30 Florida A 179 100 131 198 256 273 Wsk2.34 Florida A 179 102 131 198 256 269 Wsk2.44 Florida A 179 100 131 198 258 277 Wsk2.46 Florida A+B 179 100 131 198 258 277 CK1.3 Florida A 179 102 131 198 256 273 CK1.4 Florida A 179 102 131 198 256 273 CK1.8 Florida A+B 179 102 131 198 256 273 CK1.9 Florida A+B 179 102 131 198 256 273 CK1.5 Florida B 179 102 131 198 256 273 CK1.13 Florida A+B 179 102 131 198 256 273 CK1.14 Florida A+B 179 102 131 198 256 273 CK1.15 Florida A 179 102 131 198 256 273 CK1.16 Florida B 179 102 131 198 256 273 CK1.18 Florida A+B 179 102 131 198 256 273 CK1.23 Florida A+B 179 102 131 198 256 273 CK1.24 Florida A+B 179 102 131 198 256 273 CK1.25 Florida A+B 179 102 131 198 256 273 CK1.26 Florida A 179 102 131 198 256 273 CK1.27 Florida A+B 179 102 131 198 256 273 CK1.28 Florida A 179 102 131 198 256 273 81 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 CK1.33 Florida B 179 102 131 198 256 273 CK2.1 Florida A+B 179 102 131 198 256 273 CK2.2 Florida A+B 179 102 131 198 256 273 CK2.3 Florida B 179 102 131 198 256 273 CK2.4 Florida A+B 179 102 131 198 256 273 CK2.5 Florida A+B 179 100 131 198 258 273 CK2.7 Florida A+B 179 102 131 198 256 273 CK2.8 Florida A+B 179 102 131 198 256 273 CK2.9 Florida A+B 179 102 131 198 256 273 CK2.10 Florida A+B 179 100 131 198 258 277 CK2.11 Florida A+B 179 102 131 198 256 273 CK2.12 Florida A+B 179 102 131 198 256 273 CK2.13 Florida A+B 179 102 131 198 256 273 CK2.14 Florida A+B 179 100/102 131 198 256/258 273 CK2.15 Florida A 179 102 131 198 256 273 CK2.16 Florida A+B 179 100 131 198 258 277 CK2.17 Florida A 179 102 131 198 256 273 CK2.18 Florida A+B 179 102 131 198 256 273 CK2.19 Florida A 179 102 131 198 256 273 CK2.20 Florida A+B 179 102 131 198 256 273 CK2.21 Florida A+B 179 102 131 198 256 273 CK2.22 Florida A+B 179 102 131 198 256 273 CK2.23 Florida A+B 179 100/102 131 198 256/258 273 CK2.24 Florida A 179 102 131 198 256 273 CK2.25 Florida A+B 179 100 131 198 258 277 CK2.26 Florida A 179 100 131 198 258 273 CK2.28 Florida A 179 100/102 131 198 256 273/277 CK2.29 Florida B 179 102 131 198 256 273 82 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 CK2.30 Florida B 179 100 131 198 256 257 CK2.31 Florida A 179 102 131 198 256 273 CK2.32 Florida A+B 179 102 131 198 256 273 CK2.33 Florida A 179 102 131 198 256 273 CK2.34 Florida A+B 179 102 131 198 256 273 CK2.35 Florida A 179 100 131 198 258 273 CK2.37 Florida A 179 102 131 198 256 273 CI1.1 Hawaii B 179 98/100 129 198 254/258 253/257 CI1.2 Hawaii B 179 100 129 198 258 253/257 CI1.3 Hawaii B 179 100 129 198 258 253/257 CI1.4 Hawaii B 179 98 129 198 258 253/257 CI1.5 Hawaii B 179 98 129 198 254 253 CI1.6 Hawaii B 179 98/100 129 198/200 254/258 253/257 CI1.7 Hawaii B 179 100 129 198 258 253/257 CI1.8 Hawaii B 179 98/100 129 198 258 253/257 CI1.9 Hawaii B 179 100 129 198 258 253/257 CI1.10 Hawaii B 179 98 129 198 258 253/257 CI1.11 Hawaii B 179 100 129 198 258 253/257 CI1.12 Hawaii B 179 100 129 198/200 258 253/257 CI1.13 Hawaii B 179 98/100 129 198 254/258 253/257 CI1.14 Hawaii B 179 100 129 198 258 253/257 CI1.15 Hawaii B 179 100 129 198 258 253/257 CI1.16 Hawaii B 179 100 129 198 258 253/257 CI1.17 Hawaii B 179 98 129 198/200 258 253/257 CI1.18 Hawaii B 179 98 129 198 258 253/257 CI1.19 Hawaii B 179 98/100 129 198 254/258 253/257 CI1.20 Hawaii B 179 100 129 198 258 253/257 CI1.21 Hawaii B 179 98/100 129 198 254/258 253/257 83 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 CI1.22 Hawaii B 179 100 129 198 258 253/258 CI1.23 Hawaii B 179 98 129 198 254 253 WA1 Hawaii B 179 98/100 129 198 254/258 253/257 WA2 Hawaii B 179 98 129 198 254 253 WA3 Hawaii B 179 98/100 129 198 254/258 253/257 WA4 Hawaii B 179 98 129 198 254 253 WA5 Hawaii B 179 98 129 198 254 253 WA6 Hawaii B 179 98/100 129 198 254/258 253/257 WA7 Hawaii B 179 98 129 198 254 253 WA8 Hawaii B 179 98/100 129 198 254/258 253/257 WA9 Hawaii B 179 98 129 198 254 253 WA10 Hawaii B 179 98 129 198 254 253 WA11 Hawaii B 179 98 129 198 254 253 WA12 Hawaii B 179 98/100 129 198 254/258 253/257 WA13 Hawaii B 179 98 129 198 254 253 WA14 Hawaii B 179 98/100 129 198 254/258 253/257 WA15 Hawaii B 179 98 129 198 254 253 WA16 Hawaii B 179 98/100 129 198/200 254/258 253/257 CI2.1 Hawaii B 179 98/100 129 198/200 254/258 253/257 CI2.2 Hawaii B 179 100 129 198 258 253 CI2.3 Hawaii B 179 98 129 198 258 253 CI2.4 Hawaii B 179 98 129 198 258 253 CI2.5 Hawaii B 179 98 129 198 254 253/257 CI2.6 Hawaii B 179 98/100 129 198 258 253 CI2.7 Hawaii B 179 98 129 198 258 253 CI2.8 Hawaii B 179 98 129 198 258 253 CI2.9 Hawaii B 179 98 129 198/200 258 253/257 CI2.10 Hawaii B 179 98/100 129 198 254/258 253 84 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 CI2.11 Hawaii B 179 98 129 198/200 258 253 CI2.12 Hawaii B 179 98 129 198 254/258 253 CI2.13 Hawaii B 179 100 129 198/200 258 253/257 CI2.14 Hawaii B 179 98 129 198/200 258 253/257 CI2.15 Hawaii B 179 98/100 129 198 254/258 253 CI2.16 Hawaii B 179 98 129 198 254/258 253/257 CI2.17 Hawaii B 179 98/100 129 198 258 253 CI2.18 Hawaii B 179 98/100 129 198 258 257 CI2.19 Hawaii B 179 98/100 129 198 254/258 253/257 CI2.20 Hawaii B 179 98 129 198 258 253 CI2.21 Hawaii B 179 98 129 198 258 253/257 TMAp1 Japan B 179 98 129 198 254 253 TMAp2 Japan B 179 98 129 198 254 253 TMAp3 Japan B 179 98 129 198 254 253 TMAp4 Japan B 179 98 129 198 254 253 TMAp5 Japan B 179 98 129 198 254 253 TMAp6 Japan B 179 98 129 198 254 253 TMAp7 Japan B 179 98 129 198 254 253 TMAp8 Japan B 179 98 129 198 254 253 TMAp9 Japan B 179 98 129 198 254 253 TMAp10 Japan B 179 98 129 198 254 253 TMAp11 Japan B 179 98 129 198 254 253 TMAp12 Japan B 179 98 129 198 254 253 TMAp13 Japan B 179 98 129 198 254 253 TMAp14 Japan B 179 98 129 198 254 253 TMAp15 Japan B 179 98 129 198 254 253 TMAp16 Japan B 179 98 129 198 254 253 TMAp17 Japan B 179 98 129 198 254 253 85 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 TMAp18 Japan B 179 98 129 198 254 253 Moto Ap1 Japan B 179 98 129 198 254 253 Moto Ap2 Japan B 179 98 129 198 254 253 Moto Ap3 Japan B 179 98 129 198 254 253 Moto Ap4 Japan B 179 98 129 198 254 253 Moto Ap5 Japan B 179 98 129 198 254 253 Moto Ap6 Japan B 179 98 129 198 254 253 Moto Ap7 Japan B 179 98 129 198 254 253 Moto Ap8 Japan B 179 98 129 198 254 253 Ishi Ap1 Japan B 179 98 129 198 254 253 Ishi Ap2 Japan B 179 98 129 198 254 253 Ishi Ap3 Japan B 179 98 129 198 254 253 Ishi Ap4 Japan B 179 98 129 198 254 253 Ishi Ap5 Japan B 179 98 129 198 254 253 Ishi Ap6 Japan B 179 98 129 198 254 253 Ishi Ap7 Japan B 179 98 129 198 254 253 Ishi Ap8 Japan B 179 98 129 198 254 253 Ishi Ap9 Japan B 179 98 129 198 254 253 Ishi Ap10 Japan B 179 98 129 198 254 253 Seso Ap Lite1 Japan B 179 98 129 198 254 253 Seso Ap Lite2 Japan B 179 98 129 198 254 253 Seso Ap Lite3 Japan B 179 98 129 198 254 253 Seso Ap Lite4 Japan B 179 98 129 198 254 253 Seso Ap Lite5 Japan B 179 98 129 198 254 253 Seso Ap Lite6 Japan B 179 98 129 198 254 253 Seso Ap Lite7 Japan B 179 98 129 198 254 253 Seso Ap Dark1 Japan B 179 98 129 198 254 253 Seso Ap Dark2 Japan B 179 98 129 198 254 253 86 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 Seso Ap Dark3 Japan B 179 98 129 198 254 253 Seso Ap Dark4 Japan B 179 98 129 198 254 253 Seso Ap Dark5 Japan B 179 98 129 198 254 253 Seso Ap Dark6 Japan B 179 98 129 198 254 253 Seso Ap Dark7 Japan B 179 98 129 198 254 253 Seso Ap Dark8 Japan B 179 98 129 198 254 253 BER3 Bermuda B 179 100 129 202 254 265 BER5 Bermuda B 179 100 129 202 254 265 BER6 Bermuda B 179 100 129 202 254 265 BER9 Bermuda B 179 100 129 202 254 265 BER10 Bermuda B 179 100/104 129 202/204 254/256 265 BER11 Bermuda B 179 100 129 202 254 265 BER12 Bermuda B 179 100 129 202 254 265 BER13 Bermuda B 179 100 129 202 254 265 BER14 Bermuda B 179 104 129 204 256 265 BER15 Bermuda B 179 100 129 202 254 265 BER16 Bermuda B 179 100 129 202 254 265 BER17 Bermuda B 179 100 129 202 254 265 BER18 Bermuda B 179 104 129 204 256/258 265 BER19 Bermuda B 179 100 129 202 254 265 HERAUS1 Australia B 179 102 131 198 254 253 HERAUS2 Australia B 179 102 131 198 254 253 HERAUS3 Australia B 179 102 131 198 254 253 HERAUS4 Australia B 179 102 131 198 254 253 HERAUS5 Australia B 179 102 131 198 254 253 HERAUS6 Australia B 179 102 131 198 254 253 HERAUS7 Australia B 179 102 131 198 254 253 HERAUS8 Australia B 179 102 131 198 254 253 87 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 HERAUS9 Australia B 179 102 131 198 254 253 HERAUS10 Australia B 179 102 131 198 254 253 HERAUS11 Australia B 179 102 131 198 254 253 HERAUS12 Australia B 179 102 131 198 254 253 HERAUS13 Australia B 179 102 131 198 254 253 HERAUS14 Australia B 179 102 131 198 254 253 HERAUS15 Australia B 179 102 131 198 254 253 HERAUS16 Australia B 179 102 131 198 254 253 HERAUS17 Australia B 179 102 131 198 254 253 HERAUS18 Australia B 179 102 131 198 254 253 HERAUS19 Australia B 179 102 131 198 254 253 HERAUS20 Australia B 179 102 131 198 254 253 HERAUS21 Australia B 179 102 131 198 254 253 HERAUS22 Australia B 179 102 131 198 254 253 HERAUS23 Australia B 179 102 131 198 254 253 HERAUS24 Australia B 179 102 131 198 254 253 HERAUS25 Australia B 179 102 131 198 254 253 HERAUS26 Australia B 179 102 131 198 254 253 RSAN1 Red Sea B 179 98 129 202 254 257 RSAN2 Red Sea B 179 98 129 202 254 257 RSAN3 Red Sea B 179 98 129 202 254 257 RSAN4 Red Sea B 179 98 129 202 254 257 RSAN5 Red Sea B 179 98/100 129 200/202 254/256 257 RSAN6 Red Sea B 179 98 129 202 254 257 RSAN7 Red Sea B 179 98 129 202 254 257 RSAN8 Red Sea B 179 98/100 129 200/202 254/256 257 RSAN9 Red Sea B 179 98/100 129 200/202 254/256 257 RSAN10 Red Sea B 179 98 129 202 254 257 88 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 RSAN11 Red Sea B 179 98/100 129 200/202 254/256 257 RSAN13 Red Sea B 179 98 129 202 254 257 RSAN14 Red Sea B 179 98 129 202 254 257 RSAN15 Red Sea B 179 100 129 200 256 257 RSAN16 Red Sea B 179 98/100 129 200/202 254/256 257 RSAN17 Red Sea B 179 98/100 129 200/202 254/256 257 RSAN18 Red Sea B 179 100 129 200 256 257 RSAN21 Red Sea B 179 98/100 129 200/202 254/256 257 Eilat Ap1 Red Sea B 179 98 129 202 254 257 Eilat Ap2 Red Sea B 179 98 129 202 254 257 Eilat Ap3 Red Sea B 179 98 129 202 254 257 Eilat Ap4 Red Sea B 179 98 129 202 254 257 Eilat Ap5 Red Sea B 179 98 129 202 254 257 Eilat Ap6 Red Sea B 179 98 129 202 254 257 Eilat Ap7 Red Sea B 179 98 129 202 254 257 Eilat Ap8 Red Sea B 179 98 129 202 254 257 Eilat Ap9 Red Sea B 179 98 129 202 254 257 Eilat Ap10 Red Sea B 179 98 129 202 254 257 MXLP1 Mexico B 179 98 129 198 254 253 MXLP2 Mexico B 179 98 129 198 254 253 MXLP3 Mexico B 179 98 129 198 254 253 MXLP4 Mexico B 179 98 129 198 254 253 MXLP11 Mexico B 179 98 129 198 254 253 MXLP12 Mexico B 179 98 129 198 254 253 MXLP13 Mexico B 179 98 129 198 254 253 TLAp1 Thailand B 179 100 129 202 254 257 TLAp2 Thailand B 179 100 129 202 254 257 TLAp3 Thailand B 179 100 129 202 254 257 89 Sample Location 18S rDNA RFLP CA4.86 CA6.38 Si4 Si8 Si15 Si34 TLAp4 Thailand B 179 100 129 202 254 257 TLAp5 Thailand B 179 100 129 202 254 257 TLAp6 Thailand B 179 100 129 202 254 257 TLAp7 Thailand B 179 100 129 202 254 257 TLAp8 Thailand B 179 100 129 202 254 257 Note: sample name abbreviations and sample locations refer to figure 2.1 and table 2.4.