B]THIS IS A SUPER LONG THREAD SO BEWARE[/B] . The following paper was written during the spring semester at UB (2006). It discusses the question of whether or not clade A symbionts can be classified as shallow water algae. i am working on posting the figures and tables. The papers format is that of a scientific paper so some of it can get very tedious and meticulous (so be forwarned). The write up is of my own ability and not stolen from other sources. Where appropriate references are cited. Administration if I am violating any posting protocol please notify me and I will not allow it to happen again.
Written by Christopher A. Page
Abstract
Symbiodinium (commonly referred to as zooxanthellae) is a diverse group of unicellular algae that form crucial symbioses with many marine corals and invertebrates. They exchange photosynthates for inorganics like nitrogen, phosphorous, and carbon, with their host.
Various studies have been done to characterize this these algae, and slowly we are learning what distinguishes one clade current level of classification from another. Many studies report there is a depth distribution between clades. Clade C algae have been found to be predominant in deeper water, while clade A algaes seem to have a competitive advantage in more shallow areas (Rowan and Knowlton, 1995; LaJeunesse, 2002; Baker et. al., 1997).
This study focuses on the cladal distribution within the Caribbean coral Porites asteroides, collected from The Flower Garden Banks National Marine Sanctuary and The Dry Tortugas National Park. Analysis of DNA from samples were carried out using the Small Ribosomal Subunit (18s) of the algae, digesting it with a restriction enzyme to form a consensus banding pattern for each clade. Chloroplast genotyping was also used to further characterize the identity of the symbiont.
All samples were found to be from clade A, even though these samples were collected at depths of about 20 meters. This does not fit with the previous consensus that clade A algae are shallow water species. The outcome of this preliminary experiment could spur further study into clade A zooxanthellae taxonomy and possibly lead to further specification of classified taxa.
Introduction
A coral reef ecosystem represents an elaborate network of relationships in which one organism is tied into the fate of another. The basis for all the peripheral relationships surrounding this ecosystem are inevitably dependent on the reef building animal itself and its relationship with a group of organisms commonly referred to as zooxanthellae.
These brown colored algae form an intimate symbiotic relationship with the coral host. This occurs when the animal incorporates (through phagocytosis) the algae into its endodermal cells storing them in a vacuole like structure (Colley and Trench 1983).
Zooxanthellae are grouped into the genus Symbiodinium which incorporates a multitude of different algae (zooxanthellae are dinoflagellates) that form a mutualistic symbiosis with many different hosts types including Cnidaria, Porifera, and Mollusca (Coffroth and Santos 2005) . Symbiodinium is diverse as research has demonstrated high diversity, in biochemical structure, morphology and physiology exist within the genus (Schoenberg and Trench 1980; Govind et al. 1990; Iglesias-Prieto et al. 1991) but the defining characteristics which delineate a species have yet to be recognized.
However various techniques have helped to distinguish what separates one strain of zooxanthellae from another. The implementation of polymerase chain reaction (PCR) amplification of DNA segments and restriction enzyme digest has helped to put zooxanthellae into smaller groups or clades. Specifically the amplification of segments of gene encoding the small ribosomal subunit (18s) RNA or ssRNA (Rowan and Powers, 1991) have proven most useful to breakup what was once a large indistinguishable group of organisms, into various smaller groups.
Rowan and Powers (1991) used restriction digestion of the PCR amplified small ribosomal subunit DNA to classify zooxanthellae (Table 1). Restriction fragment length polymorphisms (RFLPââ"šÂ¬Ã¢"žÂ¢s) were seen. Zooxanthellae from the same host species showed the same RFLP pattern as other individuals within that species. Also, related algal species were found in dissimilar hosts, demonstrating that restriction digest of the 18s ribosomal subunit was a valid method for classifying Symbiodinium and that plasticity of host-symbiont relationship is present. The RFLP patterns that Rowan and Powers saw are shown in Figure 1 and will be the layout for which clade letters are assigned. This is an assumption needed throughout the paper.
Since the study done by Rowan and Powers much more has been learned of the significance of the clade delineations in zooxanthellae DNA. More specifically it has been proposed through various research endeavors that different clades of algae can be found at different depths on the reef. Rowan et. al. (1997) in his study on Montastrea spp. proposed that clades B and C represented ââ"šÂ¬Ã…"œsunââ"šÂ¬Ã‚ versus ââ"šÂ¬Ã…"œshadeââ"šÂ¬Ã‚ (which is dependent on depth of coral holobiont) specialists and that clade A was referred to as a weed, being less sensitive to stressed environments and able to proliferate where B and C could not. This claim is supported through the data he collected during a natural bleaching event. Clade C was expelled from coral tissue to a significant extent; B remained at constant numbers and A seemed to take an opportunistic role in this situation and had shown an increase in growth, indicating that the algal symbionts rather than the host animal were the determinants in bleaching response patterns (Rowan, 1997)
Upon analysis of data obtained from the western Caribbean LaJeunesse (2002) asserted that symbionts from lineage A are major components of the shallowest host communities but absent in hosts collected below 5m, also he observed that differences in depth between types within a lineage (seen here in C1 vs. C3 delineations). He had seen that C3 and C3c were predominant at greater depths especially below 5m. Here LaJeunesse used a technique that allowed for further specificity within clades (first recognized through Taq 1 digestion of 18s subunit). He attained this through sequencing of the internal transcribed spacer 2 region (ITS2).
The question that will be addressed in the following pages is whether clade A symbionts are restricted to shallow water. Special adaptations suit clade A for shallow water, in theory. Mycosporine like amino acids are known to be harbored in clade A algae and absent in the rest of the clades. These proteins help in absorbing ultra violet light that can be detrimental to the animal host (Banaszak et. al., 2000)). In shallow waters corals are exposed to higher levels of UV light.
Methods
This paper examines the cladal distribution of zooxanthellae contained in the hard coral Porites astereoides at depth. This study will consider samplings from two different sites in the Caribbean, the Flower Garden Banks National Marine Sanctuary located in the Northwest Gulf of Mexico near Texas and The Dry Tortugas National Park situated 112.63 kilometers west of Key West.
The coral samples were collected using a hammer and chisel while employing SCUBA equipment at depths of up to 70 feet in both locations. Each fragment was preserved in a solution of salt saturated DMSO or 95% ethanol and brought back to a lab where the collectors extracted the DNA contained in the polyps. The CTAB extraction protocol outlined in Coffroth (1997) was followed.
However, in the case of the Flower Garden Banks and Dry Tortuga samples the DNA was originally being harvested for the host DNA rather than the zooxanthellae DNA. Because of this the use of glass beads in the extraction process was not incorporated. Glass beads serve to lyse the algal cells open when combined with vigorous agitation, therefore exposing the zooxanthellae DNA. The actual DNA that was extracted and removed to purified samples is more specific for host DNA but should still contain an adequate amount of symbiont DNA.
The purified DNA was stored at -20 degrees Celsius. In order to replicate the zooxanthellae DNA in the samples (both Flower Garden Banks and Dry Tortugas) to a level that is more readily discerned through experimentation PCR amplification was utilized. Specifically, the small ribosomal subunit DNA encoding rRNA (ssRNA) minus 103 nucleotides at either end of the DNA fragment (Rowan and Powers, 1991) was important in establishing clade identity. The ssRNA was selected for by using zooxanthellae specific primers (ss5-ss3z (Rowan and Powers 1991b) and polymerization was carried out using Taq polymerases. PCR amplified DNA was then digested with the Taq I enzyme (procedure outlined in Rowan and Powers, 1991) to form (ideally) clade specific banding patterns (fig. 1) when run out on an agarose gel.
Zooxanthellae Chloroplast Genotyping was used to provide further resolution into the identity of the clade of each sample. Specific primers were used, 23sHYPERUP and 23sHYPERDNM13 (Santos et. al., 2003) to select for the 23s chloroplast subunit, specifically domain V in the secondary structure of the DNA for experimentation (Santos et. al., 2002). Photosensitive IRD800M13Rev (or 700) dye was used to visualize the DNA. The protocol followed that of Santos et. al. (2003). The PCR product was visualized using LI-COR NEN Global IR2 DNA sequencer system to determine the size of the variable domain V denoting cladal distribution (Fig 2). The data obtained from these processes were compiled then analyzed.
Results
The Flower Garden Banks (FGB) and Dry Tortugas yielded no results using the specific primers despite increasing the amount of DNA added to the PCR reaction soup. The possibility for human error is present, but negligible because clade standard samples amplified strongly (most of the time) which were incorporated into every run as a reference. This can be seen in figure 3. Initial extractions of the samples may not have amplified because initial extractions were for the host DNA not algal and glass beads (to disrupt algal cell walls) were not used. This may explain why small amounts of algal DNA were present.
Chloroplast genotyping is a more sensitive method and was implemented. The use of the Licor machine and its superior detection abilities allowed the small amount of zooxanthellae DNA to be amplified and visualized on a gel. Using the cladogram depicted in figure 2 the clade identity of the algal samples were determined.
However, a source of error was present in running the samples out on the Chloroplast genotyping gel. There was DNA in the negative column, which is not supposed to be there. This occurred in every one of my trials.
Forty seven samples from the Flower Garden Banks (39 adult, 8 juvenile) and 8 samples from the Dry Tortugas (all adults) were analyzed using Chloroplast genotyping. This method revealed a heavy band on the gel (for each sample) denoting Domain V of the secondary structure to be 194 base pairs in length. This means that all the samples that amplified were clade A at depths of up 21 meters.
Discussion
Although there was a possible source of error in this experiment the results from the chloroplast genotyping are considered reliable. While running the gel out we saw that there was DNA in the negative controls which is a potential source of error to the results stated above. Under each well there was highly irregular banding patterns depicted on the many gels that were ran out (see figure 4). The one thing that held constant in the samples and not in the negatives was the dark 194 band. By this, we can conclude that this outcome is reliable, although there is a slight possibility that multiple clades existed in each of the samples (that cannot be verified at this time).
This study has shown that the Porites asteroides samples yielded clade A symbionts in deep and shallow water on the reef. The results of this experiment come as highly irregular when viewed in the light of what other people have found. The establishment of Clade A symbionts as shallow water algae is well known through the works of LaJeunesse (2002), Rowan and Knowlton (1995), and Baker et al. (1997). A subset of LaJeunesse's data compared with data (subset) from this experiment can be seen in Table 2. A study on Acropora cervicornis and A. palmata yield similar results (Baker et al., 1997). Baker et al. (1997) found that A. cervicornis hosts at least two different taxa that occur predictably as a function of depth. Their results show that A. cervicornis harbor clade A in shallow water and C as depth increases. While A. palmata yields only one algal clade, that of A. Its distribution among only shallow waters holds with the notion that clade A is a shallow water symbiont. Likewise Rowan and Knowlton's (1995) data on Montastrea annularis and Montastrea foveolata agree as well. Clade A was not found in depths lower than 6m in either species (Figure 5).
The results obtained in the experiment described above suggest exactly opposite of what has been recorded in the literature. Although the small set of samples used needs to be expanded, this represents one more reason why accurate classification of zooxanthellae is needed in order to understand holobiont interactions between each other and their environment. With these vastly contrary data, much more research is needed to elucidate the differences between "shallow water A's" and "deep water A's"
Legend
Figure 1: The consensus in restriction digest fragments that Rowan and Powers witnessed with Taq I. Clade A, B, C, and D digest patterns are shown. The consensus among a large range of corals and invertebrates established ribosomal subunit analysis as useful in classification of zooxanthellae. (From Rowan and Powers, 1991)
Figure 2: Cladogram separations based on Chloroplast genotyping which gives further resolution as to the diversity within a clade. The numbers within the brackets of a clade represent the number of base pairs that make up Domain V of the 23s subunit of zooxanthellae chloroplast. Clade A incorporates 5 polymorphic lengths of the area in question. (From Santos et. al. 2003)
Figure 3: Typical results obtained in analysis of the ssRNA of zooxanthellae collected and extracted from samples from the Flower Garden Banks and the Dry Tortugas. Because samples were not extracted using glass beads the algal cells were not lysed open in a high enough frequency to produce enough DNA for analysis. The clear bands underneath wells marked as A, B, C, and D are cloned clade standards that amplify to produce its respective clade digest pattern (when digested with Taq 1). The absence of solid bands closely below wells marked 5-23 indicates that ssRNA encoding DNA was not able to be amplified, and therefore could not be analyzed with Taq 1
Figure 4: Typical results from use of the 23s Chloroplast specific primers ( Hyperup and Hyperdown) with PCR procedures. The Licor imaging system provided superior visualization of Domain V specific bands. The lanes marked ladder on the end are marked with what domain lengths they represent for reference. All samples run out correspond with an A 194 band position. Notice that contamination has caused irregular ââ"šÂ¬Ã…"œextraââ"šÂ¬Ã‚ banding in sample lanes and negative lanes (denoted by neg). The absence of the 194 band in the negative lanes indicate that the result is reliable
Figure 5: Data from Rowan and Knowltonââ"šÂ¬Ã¢"žÂ¢s (1995) study on Montastrea annularis and Montastrea foveolata. It is shown that clade A zooxanthellae are not found below 6m in either M. annularis or M. foveolata. Domination of clade C ensues in deeper depths. This is contrary to the data reported in this paper. (From Rowan and Knowlton, 1995)
Tables and Figures
Table 1: The diversity of host corals and invertebrates that Rowan and Powers (1991) included in their study are shown. From Rowan and Powers (1991)
Figure 1
Figure 2
Figure 3
Figure 4
Table 2: Results from LaJeunesse (2002)
Written by Christopher A. Page
Abstract
Symbiodinium (commonly referred to as zooxanthellae) is a diverse group of unicellular algae that form crucial symbioses with many marine corals and invertebrates. They exchange photosynthates for inorganics like nitrogen, phosphorous, and carbon, with their host.
Various studies have been done to characterize this these algae, and slowly we are learning what distinguishes one clade current level of classification from another. Many studies report there is a depth distribution between clades. Clade C algae have been found to be predominant in deeper water, while clade A algaes seem to have a competitive advantage in more shallow areas (Rowan and Knowlton, 1995; LaJeunesse, 2002; Baker et. al., 1997).
This study focuses on the cladal distribution within the Caribbean coral Porites asteroides, collected from The Flower Garden Banks National Marine Sanctuary and The Dry Tortugas National Park. Analysis of DNA from samples were carried out using the Small Ribosomal Subunit (18s) of the algae, digesting it with a restriction enzyme to form a consensus banding pattern for each clade. Chloroplast genotyping was also used to further characterize the identity of the symbiont.
All samples were found to be from clade A, even though these samples were collected at depths of about 20 meters. This does not fit with the previous consensus that clade A algae are shallow water species. The outcome of this preliminary experiment could spur further study into clade A zooxanthellae taxonomy and possibly lead to further specification of classified taxa.
Introduction
A coral reef ecosystem represents an elaborate network of relationships in which one organism is tied into the fate of another. The basis for all the peripheral relationships surrounding this ecosystem are inevitably dependent on the reef building animal itself and its relationship with a group of organisms commonly referred to as zooxanthellae.
These brown colored algae form an intimate symbiotic relationship with the coral host. This occurs when the animal incorporates (through phagocytosis) the algae into its endodermal cells storing them in a vacuole like structure (Colley and Trench 1983).
Zooxanthellae are grouped into the genus Symbiodinium which incorporates a multitude of different algae (zooxanthellae are dinoflagellates) that form a mutualistic symbiosis with many different hosts types including Cnidaria, Porifera, and Mollusca (Coffroth and Santos 2005) . Symbiodinium is diverse as research has demonstrated high diversity, in biochemical structure, morphology and physiology exist within the genus (Schoenberg and Trench 1980; Govind et al. 1990; Iglesias-Prieto et al. 1991) but the defining characteristics which delineate a species have yet to be recognized.
However various techniques have helped to distinguish what separates one strain of zooxanthellae from another. The implementation of polymerase chain reaction (PCR) amplification of DNA segments and restriction enzyme digest has helped to put zooxanthellae into smaller groups or clades. Specifically the amplification of segments of gene encoding the small ribosomal subunit (18s) RNA or ssRNA (Rowan and Powers, 1991) have proven most useful to breakup what was once a large indistinguishable group of organisms, into various smaller groups.
Rowan and Powers (1991) used restriction digestion of the PCR amplified small ribosomal subunit DNA to classify zooxanthellae (Table 1). Restriction fragment length polymorphisms (RFLPââ"šÂ¬Ã¢"žÂ¢s) were seen. Zooxanthellae from the same host species showed the same RFLP pattern as other individuals within that species. Also, related algal species were found in dissimilar hosts, demonstrating that restriction digest of the 18s ribosomal subunit was a valid method for classifying Symbiodinium and that plasticity of host-symbiont relationship is present. The RFLP patterns that Rowan and Powers saw are shown in Figure 1 and will be the layout for which clade letters are assigned. This is an assumption needed throughout the paper.
Since the study done by Rowan and Powers much more has been learned of the significance of the clade delineations in zooxanthellae DNA. More specifically it has been proposed through various research endeavors that different clades of algae can be found at different depths on the reef. Rowan et. al. (1997) in his study on Montastrea spp. proposed that clades B and C represented ââ"šÂ¬Ã…"œsunââ"šÂ¬Ã‚ versus ââ"šÂ¬Ã…"œshadeââ"šÂ¬Ã‚ (which is dependent on depth of coral holobiont) specialists and that clade A was referred to as a weed, being less sensitive to stressed environments and able to proliferate where B and C could not. This claim is supported through the data he collected during a natural bleaching event. Clade C was expelled from coral tissue to a significant extent; B remained at constant numbers and A seemed to take an opportunistic role in this situation and had shown an increase in growth, indicating that the algal symbionts rather than the host animal were the determinants in bleaching response patterns (Rowan, 1997)
Upon analysis of data obtained from the western Caribbean LaJeunesse (2002) asserted that symbionts from lineage A are major components of the shallowest host communities but absent in hosts collected below 5m, also he observed that differences in depth between types within a lineage (seen here in C1 vs. C3 delineations). He had seen that C3 and C3c were predominant at greater depths especially below 5m. Here LaJeunesse used a technique that allowed for further specificity within clades (first recognized through Taq 1 digestion of 18s subunit). He attained this through sequencing of the internal transcribed spacer 2 region (ITS2).
The question that will be addressed in the following pages is whether clade A symbionts are restricted to shallow water. Special adaptations suit clade A for shallow water, in theory. Mycosporine like amino acids are known to be harbored in clade A algae and absent in the rest of the clades. These proteins help in absorbing ultra violet light that can be detrimental to the animal host (Banaszak et. al., 2000)). In shallow waters corals are exposed to higher levels of UV light.
Methods
This paper examines the cladal distribution of zooxanthellae contained in the hard coral Porites astereoides at depth. This study will consider samplings from two different sites in the Caribbean, the Flower Garden Banks National Marine Sanctuary located in the Northwest Gulf of Mexico near Texas and The Dry Tortugas National Park situated 112.63 kilometers west of Key West.
The coral samples were collected using a hammer and chisel while employing SCUBA equipment at depths of up to 70 feet in both locations. Each fragment was preserved in a solution of salt saturated DMSO or 95% ethanol and brought back to a lab where the collectors extracted the DNA contained in the polyps. The CTAB extraction protocol outlined in Coffroth (1997) was followed.
However, in the case of the Flower Garden Banks and Dry Tortuga samples the DNA was originally being harvested for the host DNA rather than the zooxanthellae DNA. Because of this the use of glass beads in the extraction process was not incorporated. Glass beads serve to lyse the algal cells open when combined with vigorous agitation, therefore exposing the zooxanthellae DNA. The actual DNA that was extracted and removed to purified samples is more specific for host DNA but should still contain an adequate amount of symbiont DNA.
The purified DNA was stored at -20 degrees Celsius. In order to replicate the zooxanthellae DNA in the samples (both Flower Garden Banks and Dry Tortugas) to a level that is more readily discerned through experimentation PCR amplification was utilized. Specifically, the small ribosomal subunit DNA encoding rRNA (ssRNA) minus 103 nucleotides at either end of the DNA fragment (Rowan and Powers, 1991) was important in establishing clade identity. The ssRNA was selected for by using zooxanthellae specific primers (ss5-ss3z (Rowan and Powers 1991b) and polymerization was carried out using Taq polymerases. PCR amplified DNA was then digested with the Taq I enzyme (procedure outlined in Rowan and Powers, 1991) to form (ideally) clade specific banding patterns (fig. 1) when run out on an agarose gel.
Zooxanthellae Chloroplast Genotyping was used to provide further resolution into the identity of the clade of each sample. Specific primers were used, 23sHYPERUP and 23sHYPERDNM13 (Santos et. al., 2003) to select for the 23s chloroplast subunit, specifically domain V in the secondary structure of the DNA for experimentation (Santos et. al., 2002). Photosensitive IRD800M13Rev (or 700) dye was used to visualize the DNA. The protocol followed that of Santos et. al. (2003). The PCR product was visualized using LI-COR NEN Global IR2 DNA sequencer system to determine the size of the variable domain V denoting cladal distribution (Fig 2). The data obtained from these processes were compiled then analyzed.
Results
The Flower Garden Banks (FGB) and Dry Tortugas yielded no results using the specific primers despite increasing the amount of DNA added to the PCR reaction soup. The possibility for human error is present, but negligible because clade standard samples amplified strongly (most of the time) which were incorporated into every run as a reference. This can be seen in figure 3. Initial extractions of the samples may not have amplified because initial extractions were for the host DNA not algal and glass beads (to disrupt algal cell walls) were not used. This may explain why small amounts of algal DNA were present.
Chloroplast genotyping is a more sensitive method and was implemented. The use of the Licor machine and its superior detection abilities allowed the small amount of zooxanthellae DNA to be amplified and visualized on a gel. Using the cladogram depicted in figure 2 the clade identity of the algal samples were determined.
However, a source of error was present in running the samples out on the Chloroplast genotyping gel. There was DNA in the negative column, which is not supposed to be there. This occurred in every one of my trials.
Forty seven samples from the Flower Garden Banks (39 adult, 8 juvenile) and 8 samples from the Dry Tortugas (all adults) were analyzed using Chloroplast genotyping. This method revealed a heavy band on the gel (for each sample) denoting Domain V of the secondary structure to be 194 base pairs in length. This means that all the samples that amplified were clade A at depths of up 21 meters.
Discussion
Although there was a possible source of error in this experiment the results from the chloroplast genotyping are considered reliable. While running the gel out we saw that there was DNA in the negative controls which is a potential source of error to the results stated above. Under each well there was highly irregular banding patterns depicted on the many gels that were ran out (see figure 4). The one thing that held constant in the samples and not in the negatives was the dark 194 band. By this, we can conclude that this outcome is reliable, although there is a slight possibility that multiple clades existed in each of the samples (that cannot be verified at this time).
This study has shown that the Porites asteroides samples yielded clade A symbionts in deep and shallow water on the reef. The results of this experiment come as highly irregular when viewed in the light of what other people have found. The establishment of Clade A symbionts as shallow water algae is well known through the works of LaJeunesse (2002), Rowan and Knowlton (1995), and Baker et al. (1997). A subset of LaJeunesse's data compared with data (subset) from this experiment can be seen in Table 2. A study on Acropora cervicornis and A. palmata yield similar results (Baker et al., 1997). Baker et al. (1997) found that A. cervicornis hosts at least two different taxa that occur predictably as a function of depth. Their results show that A. cervicornis harbor clade A in shallow water and C as depth increases. While A. palmata yields only one algal clade, that of A. Its distribution among only shallow waters holds with the notion that clade A is a shallow water symbiont. Likewise Rowan and Knowlton's (1995) data on Montastrea annularis and Montastrea foveolata agree as well. Clade A was not found in depths lower than 6m in either species (Figure 5).
The results obtained in the experiment described above suggest exactly opposite of what has been recorded in the literature. Although the small set of samples used needs to be expanded, this represents one more reason why accurate classification of zooxanthellae is needed in order to understand holobiont interactions between each other and their environment. With these vastly contrary data, much more research is needed to elucidate the differences between "shallow water A's" and "deep water A's"
Legend
Figure 1: The consensus in restriction digest fragments that Rowan and Powers witnessed with Taq I. Clade A, B, C, and D digest patterns are shown. The consensus among a large range of corals and invertebrates established ribosomal subunit analysis as useful in classification of zooxanthellae. (From Rowan and Powers, 1991)
Figure 2: Cladogram separations based on Chloroplast genotyping which gives further resolution as to the diversity within a clade. The numbers within the brackets of a clade represent the number of base pairs that make up Domain V of the 23s subunit of zooxanthellae chloroplast. Clade A incorporates 5 polymorphic lengths of the area in question. (From Santos et. al. 2003)
Figure 3: Typical results obtained in analysis of the ssRNA of zooxanthellae collected and extracted from samples from the Flower Garden Banks and the Dry Tortugas. Because samples were not extracted using glass beads the algal cells were not lysed open in a high enough frequency to produce enough DNA for analysis. The clear bands underneath wells marked as A, B, C, and D are cloned clade standards that amplify to produce its respective clade digest pattern (when digested with Taq 1). The absence of solid bands closely below wells marked 5-23 indicates that ssRNA encoding DNA was not able to be amplified, and therefore could not be analyzed with Taq 1
Figure 4: Typical results from use of the 23s Chloroplast specific primers ( Hyperup and Hyperdown) with PCR procedures. The Licor imaging system provided superior visualization of Domain V specific bands. The lanes marked ladder on the end are marked with what domain lengths they represent for reference. All samples run out correspond with an A 194 band position. Notice that contamination has caused irregular ââ"šÂ¬Ã…"œextraââ"šÂ¬Ã‚ banding in sample lanes and negative lanes (denoted by neg). The absence of the 194 band in the negative lanes indicate that the result is reliable
Figure 5: Data from Rowan and Knowltonââ"šÂ¬Ã¢"žÂ¢s (1995) study on Montastrea annularis and Montastrea foveolata. It is shown that clade A zooxanthellae are not found below 6m in either M. annularis or M. foveolata. Domination of clade C ensues in deeper depths. This is contrary to the data reported in this paper. (From Rowan and Knowlton, 1995)
Tables and Figures
Table 1: The diversity of host corals and invertebrates that Rowan and Powers (1991) included in their study are shown. From Rowan and Powers (1991)
Figure 1
Figure 2
Figure 3
Figure 4
Table 2: Results from LaJeunesse (2002)
, along with data obtained from this study
. In LaJeunesseââ"šÂ¬Ã¢"žÂ¢s study samples were taken from two different locations Puerto Morelos, Mexico and Lee Stocking Island, Bahamas. The trend observed by LaJeunesse was that clade A symbionts (analysis of ITS2 regions) were not found below 4 meters in depth among the many sampled. The data obtained in this study found clade A at depths of >20m based on chloroplast genotyping (Table on left from LaJeunesse, 2002).
Figure 5
Work Cited
Nansi J. Colley and R.K. Trench. 1983. Selectivity in phagocytosis and persistence of symbiotic algae by the scyphistoma stage of the jellyfish Cassiopeia xamachana, Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 219, No. 1214,
pp. 61-82
Anastazia T. Banaszak, Todd C. LaJeunesse, Robert K. Trench. 2000, The synthesis of mycosporine-like amino acids (MAAs) by
cultured, symbiotic dinoflagellates, Journal of Experimental Marine Biology and Ecology. vol. 249 pp219-233
NS Govind, SJ Roman, R Iglesias-Prieto, K Trench, EL Triplett, BB Prezelin, 1990, An analysis of the light harvesting peridinin-chlorophyll a-proteins from dinoflagellates by immunoblotting techniques, Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 240, pp.187-195
R Iglesias-Prieto, NS Govind, RK Trench, 1991, Apoprotein Composition and Spectroscopic Characterization of the Water-Soluble Peridinin--Chlorophyll a--Proteins from Three Symbiotic Dinoflagellates, Proceedings: Biological Sciences, Vol. 246, No. 1317, pp. 275-283
D.K. Schoenberg and R.K. Trench, 1980, Genetic Variation in Symbiodinium (=Gymnodinium) microadriaticum Freudenthal, and Specificity in its Symbiosis with Marine Invertebrates. I. Isoenzyme and Soluble Protein Patterns of Axenic Cultures of Symbiodinium microadriaticum, Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 207, No. 1169, pp.405-427
Rob Rowan and Dennis A. Powers, 1991a, A Molecular Genetic Classification of Zooxanthellae and the Evolution of Animal-Algal Symbioses,
Science, vol.251, pp.1348-1351
Rob Rowan and Dennis A. Powers, 1991b, Molecular genetic identification of symbiotic dinoflagellates (zooxanthellae), Marine ecology progress series. Oldendorf [MAR. ECOL. (PROG. SER.).]. Vol. 71, no. 1, pp. 65-73. 1991.
Rob Rowan, 1997, Diversity and Ecology of Zooxanthellae on Coral Reefs,
Journal of Phycology [J. Phycol.]. Vol. 34, no. 3, pp. 407-417
Todd C. LaJeunesse, 2002, Diversity and community structure of symbiotic dinoflagellates from Caribbean coral reefs,
Published online: Springer-Verlag
Scott R. Santos, Carla Guitierezz-Rodriguez, and Mary Alice Coffroth, 2003, Phylogenetic Identification of Symbiotic Dinoflagellates via Length Heteroplasmy in Domain V of Chloroplast Large Subunit (cp23s)-Ribosomal DNA sequences, Marine Biotechnology, vol. 5, pp.130-140
Scott R. Santos, Derek J. Taylor, Robert A. Kinzie III, Kazuhiko Sakai, and Mary Alice Coffroth, 2002, Evolution of length variation and heteroplasmy in the chloroplast rDNA of symbiotic dinoflagellates (Symbiodinium, Dinophyta) and a novel insertion in the universal core region of the large subunit rDNA, Phycologia, Volume 41, pp.311-318
Andrew C. Baker, Rob Rowan, and Nancy Knowlton, 1997, Symbiosis ecology of two Caribbean Acroporid Corals, Proceedings of the 8th International Coral Reef Symposium; Vol. 2, pp.1295-1300
Coffroth and Santos, 2005, Genetic diversity of Symbiotic Dinoflagellates in the Genus Symbiodinium, Protist, Vol. 1 published online
MA Coffroth, SR Santos, 1997, Early ontogeny of Zooxanthellae-coral-symbiosis-a preliminary report. Am Zoology vol. 37, 11A
Rob Rowan and Nanci Knowlton, 1995, Intraspecific Diversity and Ecological Zonation in Coral-Algal Symbiosis, Proceedings of the National Academy of Sciences of the United States of America, Vol. 92 No. 7, pp.2850-2853
[
Figure 5
Work Cited
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