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1 The Defense Mechanisms and Reproductive Barriers of Spirobranchus giganteus Meg Rippy & Jen Kelleher Abstract: Spirobranchus giganteus is a species or species complex of serapulid polychaetes that lives on hermatypic coral. Its defense mechanisms and reproductive barriers were studied in Opunohu Bay, Moorea, French Polynesia, November 2004. The removal of opercular spines, which have been considered a defense mechanism, was observed to have no effect on Christmas tree worm (CTW) fitness. Interestingly, however, spine regrowth was observed, and more CTW’s exhibited this behavior over time. We estimate that full spine regrowth will take 182 days. In the part of this experiment devoted to reproductive barriers, three surveys were conducted. S. giganteus was found to aggregate by color within, but not between, coral heads and there was no association found between cohort age and CTW color. These patterns are consistent with the hypothesis that CTW color morphs represent different species, assuming CTW’s reproduce at the same time and are small-scale broadcast spawners. Color morph reproductive compatibility was examined directly by crossing three color morphs, where the larvae produced were observed for 24 hours. The development of these larvae suggests that the Y and RW color morphs are the same species, the RW and WPT color morphs are different species, and the Y and WPT color morphs are different species that don’t exhibit complete reproductive incompatibility. Introduction: Spirobranchus giganteus is a species or species complex of serapulid polychaetes that exist in a symbiotic relationship with hermatypic corals (Toonen, 20021). These polychaetes, also known as Christmas tree worms (CTW’s), secrete calcium carbonate tubes and are believed to induce the corals they inhabit to grow up around them. The worm itself has a double spiral feeding structure that comes in many different colors. They are responsive to tactile stimuli and very rapidly retreat into their tubes when approached. On the operculum of the tube, supposedly to protect the worm from predators, is a large calcium carbonate spine. This spine could be instrumental in protection against butterfly fish such as Chaeteodon lunula, which as juveniles have a gut content that is 62.3% polychaetes (Harmelin-Vivien, 1988). One of the several purposes of this study is to examine the function of the CTW opercular spine by observing worms that have had their spines removed and relating their survival over time to worms that have not been altered. We hypothesize that when the opercular spine of S. giganteus is cut it will be preyed upon more frequently than S. giganteus with intact opercular spines. Although the adult stage of S. giganteus is interesting in its own right, it is the larvae of CTW’s that will be the main focus of this study. As was previously stated, S. giganteus may be a species complex with different color morphologies representing different species (Marsden, 2002, as cited in Toonen, 2002). Spirobranchus polycerus, a sister species to S. giganteus has been shown to be such a complex, where larvae produced by crossing different morphs do not develop properly (Toonen, 20021). This form of barrier is postzygotic and called hybrid inviability. It is also possible that prezygotic barriers could be present in S. 1 Toonen, R. J. 2000. Porites and “Christmas Tree Worms”. Reefs.org Article (http://www.reefs.org/library/article/r_toonen21.html) 2 giganteus, where the sperm and eggs of different morphs are completely incompatible and no larvae are produced at all (Palumbi, 1994). In this study three color morphs (yellow <Y>, red/white <RW> and white/pink/tan <WPT>) will be crossed in order to check for the presence of these two types of reproductive barriers. We hypothesize that cross color mating will either produce no larvae, or larvae that do not progress through the normal morphological growth stages. If it is assumed that the Y, RW, and WPT color morphs of S. giganteus are actually different species, three hypotheses concerning the distributions of these morphs on different coral heads can be made. First, individual CTW colors should group together on any one coral head because these organisms are broadcast spawners that mate most with their neighbors (Cronin, 2002). If closely associated CTW’s were different species (ie different colors), then, using the biological species concept’s definition of a species, they would not be capable of mating. In this light it would be beneficial for a CTW to be positioned next to a morph of the same color so that it could mate effectively. A prediction related to this, but on the scale of an entire reefal area, is that the color composition of CTW’s between coral heads will vary because larvae should settle near same color conspecifics for mating purposes. This would lead to a patchy distribution of color morphs between corals as well as among them. A further hypothesis concerning the distribution of CTW color morphs involves the association between the size of an individual and its color. If different colors represent different species, larvae from a red-red mating should be ready to settle in a particular area at a slightly different time than larvae from a blue-blue mating, assuming the matings occur at different times. Because CTW tube diameter is indicative of both the size and age of an individual, it should be possible to scan recruitment pulses over time and observe whether they contain a single color morph or not (Nishi and Nishihira, 1996, as cited in Toonen, 2002; Cronin, 2002). The presence of many different colors in one recruitment pulse would indicate either that S. giganteus color morphs do not represent separate species or that all colors of CTW’s, whether they are different species or not, tend to spawn at the same time. The presence of only one color in a recruitment pulse would suggest that different S. giganteus color morphs are separate species. Materials and Methods Study Site: This study was conducted from November 13th 2004 to December 6th 2004 at Public Beach in Opunohu Bay, Moorea, French Polynesia. At Public Beach two sites were used. The first site was the crest patch, which is right inside the crest. The crest patch was used for distribution, and age estimation studies. The crest patch was chosen for these tests because the Porites Lobada coral heads found there were large and contained hundreds of CTW’s. The second site was the fringing patch, which is farther inside the lagoon than the crest patch, next to the fringing reef. We chose this site for the opercular spine removal study and the spine regrowth 3 study because the Porites Lobada coral heads there were smaller, flatter, and contained fewer CTW’s, making cage placement and spine measurement easier. The Effect of Opercular Spine Removal on the fitness of S. giganteus: At the fringing patch site we chose six different Porites Lobata coral heads containing at least 8 CTW’s, two of which were close to each other, for use in our study on the effect spine removal has on CTW’s. The two closely positioned individuals, one which had its spine removed and one where the spine was left intact, were caged together. The cut caged CTW was used as a control for spine removal, where the death of this worm would indicate that the removal process itself causes CTW mortality. The uncut caged worm was a control for the cage, where increases or decreases in the death rates of these CTW’s relative to the death rates of uncut uncaged individuals would indicate an effect of the cage on survival. Six uncaged CTW’s, three of which had their spines cut, were also observed on each coral head in order to compare CTW survival without predators (caged) to survival where predation was possible. Theoretically, if opercular spines increase survival rates, uncaged cut CTW’s will die over time and uncaged uncut CTW’s will survive. Only if caged cut CTW’s have lower mortalities than uncaged cut individuals can this difference in survival be attributed to predation. The six replicate coral heads used in this experiment were observed every other day for three and a half weeks, taking note of whether or not the caged/uncaged/cut/uncut CTW’s were alive. Spine Regrowth in Spirobranchus giganteus The number of CTW’s that had regrown their spines on each of the six coral heads used in the spine removal experiment discussed above was tallied in order to examine how continuous removal affected spine growth over time. These counts were taken approximately every other day, from November 17th to December 6th. Spine length was also measured for one CTW on each of these six coral heads over a two day, three day, five day and 21 day period. This data was used in context with the average length of CTW spines, calculated by averaging the spine length of five CTW’s on three coral heads, in order to estimate the average time it takes a CTW to grow a full spine. Test for Cross Morph Incompatability: One male and one female of each color (Y, RW, and WPT) were used in reproductive crosses designed to examine CTW color morph reproductive compatibility. Reproductive incompatibility, in the form of larval death, larval mutation, or larval absence, was taken as evidence supporting the hypothesis that CTW color morphs represent different species. The CTW’s were forced to spawn in a lab using different techniques; changing water temperature, a drastic change in light, splitting open the tube, and dissecting the worm. The eggs and sperm of each color morph were mixed with each of the other color morphs, as well as their own, for a total of 9 crosses. Each cross was performed two times. 4 Y male R/W male WPT male Y female YmxYf R/W m x Y f WPT m x Y f R/W female Y m x R/W f R/W m x R/W f WPT m x R/W f WPT female Y m x WPT f R/W m x WPT f WPT m x WPT f Thirty minutes after mixing, the fertilized eggs were washed and placed into a glass beaker so that there was a concentration of about one egg per milliliter. The development process was observed for 24 hours in each of the crosses. At 30 minutes we checked for evidence of fertilization and at, 60 minutes, 90 minutes, 16 hours, 20 hours, 21 hours & 24 hours the larvae were observed for developmental changes under a microscope. These time increments were chosen based on a study performed by Shawn Cronin in 2002. Distribution of CTW’s: At the Public Beach crest patch reef, CTW distribution was observed for 22 random Porites Lobada coral heads. For each coral head the total number of CTW’s was counted and the total number of Y, RW, and WPT individuals was noted. The sum of these three colors was subtracted from the total number of CTW’s observed in order to quantify the number of “other” color morphs on the coral heads. These four color morph totals were used in a Pearson chi-squared test to examine whether or not the distribution of CTW color morphs changes from coral head to coral head. This addresses the hypothesis that S. giganteus is a species complex with color morphs representing separate species that aggregate together over an entire reef section. In order to test the hypothesis that CTW’s aggregate within coral heads as well as between them, 5 quadrats on 11 individual coral heads were surveyed for CTW color. The total number of Y, RW & WPT CTW’s on each of these corals was also counted in order to determine prior probabilities for these colors. A binomial distribution was used to determine whether or not observing two or more Y, RW or WPT color morphs in any given quadrat was likely, based on these priors. The idea here was to determine whether or not aggregations of like color morphs occurred more often than would be expected by chance. To test the hypothesis that, because different color morphs represent different species, larvae will settle in temporal waves of same color cohorts, the tube diameter of five Y, RW, WPT, blue and black/white color morphs, respectively, was measured for each of five coral heads. Tube diameter was converted to CTW age using the 0.2mm/year growth rate constant cited by Rob Toonan (Toonan, 2002). The resulting distributions of CTW age were compared graphically in terms of color morph, and then analyzed using a Pearson chi-squared test. Results The Effect of Opercular Spine Removal on the fitness of S. giganteus: 5 The percent of CTW’s, both cut and uncut, caged and uncaged, that survived over time is shown in Graph 1. Survival over the 22 day period was 100 percent for all treatments. Spine Regrowth in Spirobranchus giganteus: The change in the average size of spines regrown over time is depicted for 30-year-old CTW’s in Graph 2. The relationship is a power function (Table 1., P=0.00) that, when converted to a linear form, can be represented by the equation y = 0.132x + 0.001. Using this equation and the average spine size of a 30-year-old CTW (4.7cm), the time required for full spine growth was estimated to be 182 days. The number of CTW’s that regrew spines over the entirety of the opercular spine removal experiment is depicted in Graph 3. Overall this trend is linear (Graph 4 & Table 2., P = 0.00), though there was a pronounced valley on the ninth day of observation. Overall, more CTW’s were regrowing their spines by the end of the experiment than at the beginning. Test for Cross Morph Incompatibility: For the pure Ym x Yf, RWm x RWf and WPTm x WPTf color morph crosses the following developmental stages were observed: Time after release Observed Characteristics 15 minutes Unfertilized egg. Sperm swimming, not attacking 30 minutes Sperm attacking eggs 60 minutes Sperm attacking eggs 90 minutes Sperm blocked by a fertilization wall 16 hrs Cell wall present (division occurring) 20 hrs Proto-trochophore formed (cilia not yet present) 24 hrs Trochophore ciliated; swimming observed 2-5 days (Ym x Yf only) Trochophore refined; reduced stomach and elongated cilia 5-8 days (Ym x Yf only) Metatrochopore observed For both replicates of the WPTm x RWf and RWm x WPTf crosses, normal larval development was not observed. The first developmental problems occurred at 90 minutes and all involved abnormal budding of the larvae into segments. Developmental abnormalities were also observed in 24-hour-old motile larvae, ranging from elongated body plans with few cilia to tear drop shaped forms with cilia concentrated on their pointed ends. The Y x WPT color morph crosses, unlike the RW x WPT crosses, produced different larval forms depending on the direction of the cross. When the male was Y and the female was WPT, trochophore larvae were produced with two patches of cilia that covered ½ of the body. When the female was Y, however, two different results were obtained. In one replicate a mixture of mutant and normal trochophore larvae were 6 produced. These mutants were oddly shaped and had breaks in their cilia where their outer walls were not circular. In the second replicate no larvae were produced at all. Of the matings involving different color morphs only the Y x RW crosses produced no mutant larvae. It is significant, however, that the first two reciprocal crosses for these colors produced no larvae at all, while the second two produced normal trochophores by the 24-hour mark. Distribution of CTW’s: The distribution of CTW color morphs between coral heads is shown in Graph 5 and Graph 6. Graph 6 excludes the ‘other’ category, which contains all CTW’s that were not Y, RW, or WPT. Both graphs show that the total number of CTW’s per coral varies between coral heads. The variation in the relationship between the number of CTW’s in each color morph category, however, is only significant when the ‘other’ category is included in the analysis (Table 3., P=6.9x10^-6). When the ‘other’ category is excluded, no significant variation in CTW color morph distribution is observed between coral heads. (Table 4., P= 0.29). Although the CTW color morph distribution between coral heads showed insignificant variation for Y, RW, and WPT morphs, the CTW color morph distribution within a single coral head, for these same color morphs, showed marginally significant variation. The average probability of a CTW being found next to its same color conspecific by chance was 0.10 for Y, 0.06 for RW, and 0.13 for WPT. Graphs 7 and 8, which depict the number of instances where Y, RW, or WPT color morphs were observed alone or with same color conspecifics, respectively, show that for all three color morphs more CTW’s were found in same color groupings than not. The third distributional study concerning the relationship between CTW cohort age and the color morphs observed in that cohort, had a highly insignificant P value of 0.98 (Table, 5). This is represented in Graph 9, which shows similar age distributions for each color morph, where few CTW’s were observed under twenty or over forty years old. Fifty-eight percent of CTW’s over all the distributions (Y, RW, WPT, blue and black/white) were between these two ages. Discussion Opercular spine removal appears to have no effect on CTW fitness over relatively short periods of time. Because there was no mortality observed for any of the cut, uncut, caged or uncaged treatments, it is impossible to say whether or not opercular spines protect CTW’s from predation. The numerous cases of spine regrowth, however, indicate that CTW spines serve an important purpose for the CTW’s, even if the nature of that purpose has yet to be defined. It is notable that the initial speed of spine growth was rapid and that it decreased over time, suggesting that more effort is put into the initial recovery of the spine (Graph 2). It may be that large spine size is unimportant relative to spine presence. 7 Another indication that CTW spines serve an important purpose is that the number of CTW’s that regrew spines increased over time (Graph 3). This could suggest that the importance of these spines is related to protection, if the increased spine regrowth was stimulated by our continual spine removal and represented a stress-induced defense mechanism. It is also possible, however, that external factors other than direct human interference caused the increase in the number of CTW’s that regrew spines over time. It is notable that there was one day of observation where fewer CTW’s regrew spines than the day before. Temperature, rainfall, wind speed, and wave height were examined in the attempt to explain this reduction in regrowth, but none of these factors shared an obvious graphical relationship with spine regrowth over time (Graph 10). The second part of our study moved beyond opercular spine regrowth and focused on the distribution of adult CTW’s on the basis of color, where different colors were equated with different species. The fact that the distribution of Y, RW and WPT CTW’s on any one coral head was found to be incredibly similar to the distribution of these same morphs on other coral heads indicates that if CTW’s aggregate by color morph/species they do so on a scale smaller than the one examined here. We believe that the significant P value for the between coral head distribution study, when the ‘other’ category was included, is an artifact of lumping over 29 different CTW color morphs into one group. This lumping confounds the number of total worms with the number of those worms that were different colors, making the CTW composition of coral heads examined appear more different then they really were. The idea that CTW’s aggregate on a smaller scale than a coral head community, suggested in the paragraph above, was shown to be likely when the spatial distribution of CTW color morphs was examined on individual coral heads. The marginally significant P values for color morph aggregation within a coral head suggest that some form of preference for same color conspecifics is exhibited by CTW larvae. This preference may well be due to mating incompatibility between different color morphs, but it could also be due to other factors. An example of this would be gregarious settlement, assuming the larval cohort settling contained a large number of RW morphs. In such a situation many RW morphs would be clumped together, but this clumping would be due to a preference for conspecifics in general (perhaps because CTW’s are broadcast spawners) not a preference for conspecifics based on reproductive incompatibility between color morphs. On the subject of larval cohorts, their color morph composition, and reproductive incompatibility between color morphs, it is important to note that different color morphs do not exhibit statistically different cohort ages (Table 5). This, however, does not necessarily suggest that different CTW color morphs are not separate species. It is possible that same color crosses produce larval cohorts at the same time, which would mix color morphs together that would otherwise be in species specific groups. In such a situation age would be a poor method for distinguishing between cohorts. Age estimation has another flaw in that it is derived from tube diameter measurements, which increase at a rate of 0.2mm/year. This means that even if the mating times of different color morphs were separated by months, it would be difficult to distinguish between the larval cohorts produced because of the incredibly small differences in their tube diameters. 8 The purpose of the CTW color morph distribution and age/cohort studies was to test predictions based on the hypothesis that different CTW color morphs represent different species. Larval crosses performed in the lab tested this hypothesis directly, through the examination of larval development. The fact that RW x WPT crosses produced entirely mutant larvae suggests that these two color morphs represent different species. The presence of several larvae at 90 minutes that had budded into multiple segments and arrested development is particularly interesting because it provides definitive evidence of fatality due to reproductive incompatibilities. This cross also produced mutants that were ciliated and swimming. For these individuals it is entirely possible that, although they appear odd, they could grow up, settle normally, and become fertile adults. In this light, although it is likely that RW and WPT color morphs exhibit postzygotic reproductive barriers, we cannot be certain. Unlike the RW x WPT cross, which only produced mutant larvae, the RW x Y cross produced either normal or no larvae. Because both directions of the cross produced both results, it is believed that RW x Y matings result in viable offspring. The Y female used in the RWm x Yf cross that produced no larvae is thought to have been largely infertile with few immature eggs. This is corroborated by the results of a WPTm x Yf cross, where when the same Yf was used no larvae were produced and when a different Yf was used viable larvae were produced. The absence of larvae in the Ym x RWf cross was likely caused by the numerous dinoflagellates and ciliates observed in the seawater these larvae should have inhabited. All together these observations suggest that RW and Y CTW’s produce normal larvae and are the same species. Assuming that RW and Y CTW’s are the same species, and RW and WPT CTW’s are different species, one can logically conclude that Y and WPT CTW’s are different species as well. This logic is supported by the results of the WPT x Y CTW crosses performed, where the WTPf x Ym cross produced mutant ciliated forms and the WPTm x Yf cross produced a mixture of normal and mutant ciliated forms. It is obvious that there are no prezygotic barriers between these color morphs, but the presence of the mutants makes postzygotic barriers a possibility. It is particularly interesting that one of the reciprocal crosses produced more mutants than the other, because this indicates that in some cases color morph cross direction can determine reproductive outcome. The fact that one cross produced both mutants and normal larvae is also interesting because it suggests that Y and WPT color morphs are not completely reproductively isolated. Acknowledgements: Big special thanks to Jared for installing all of the cages used in the opercular spine removal study. Thanks to crazy Pete, PTR, Jared, Dawn, and everyone else for helping us collect CTW’s. And to PTR and Giacomo, we can’t thank you enough for the opportunity to do research someplace other than in Santa Cruz, which is currently experiencing a cold, wet, rainy winter. 9 References Cronin, S. 2002. Spatial Distribution and Larval Biology of Spirobranchus giganteus. Bio 162 Unpublished Harmelin-Vivian, M; Bouchon-Navaro, Y; Galzin, R .1988. Patterns of Distribution of Herbiverous Reef Fishes in French Polynesia. Proc. Sixth Intt. Coral Reefs Symp., Townsville,: 41., - Vol 18 Palumbi S, R. 1994. Genetic Divergence, Reproductive Isolation, and Marine Speciation. Annu. Rev. Ecol. Syst. 25:547-72 Toonen, R. J. 2002. Invertebrate Non-Column: Christmas Tree Worms. Advanced Aquarist’s Online Magazine. (http://www.advanceaquarist.com/issues/mar2002/toonen.htm) Toonen, R. J. 2000. Porites and “Christmas Tree Worms”. Reefs.org Article (http://www.reefs.org/library/article/r_toonen21.html) 10 Graph 1. Percentage of CTW’s that are alive and cut, alive and uncut, dead and cut or dead and uncut, as grouped by cage treatment. 120 100 Value 80 60 40 ALIVEC ALIVEUC DEADC DEADUC 20 0 no yes CAGED Graph 2: The average length of CTW spines regrown over time 0.5 Spine Length 0.4 0.3 0.2 0.1 0.0 0 50 100 Day 150 200 Table 1: Regression analysis for the linearized graph of average regrown CTW spine length over time. Source Regression Residual Sum-ofSquares df MeanSquare F-ratio P 0.042 1 0.042 425.201 0.000 0.000 3 0.000 11 Graph 3: The number of CTW’s exhibiting spine regrowth over time # CTW Exhibiting Spine Regrowth 3.5 3.0 2.5 2.0 CORAL 1.5 1 2 3 4 5 6 1.0 0.5 0.0 0 5 10 15 20 25 Day Graph 4: The linear trend exhibited by the number of CTW’s exhibiting spine regrowth over time # CTW Exhibiting Spine Regrowth 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 5 10 15 20 25 Day Table 2: Linear regression analysis for the trend exhibited by the number of CTW’s exhibiting spine regrowth over time Source Regression Residual Sum-ofSquares df MeanSquare 90.796 1 90.796 27.787 58 0.479 F-ratio 189.520 P 0.000 12 Graph 5: CTW color morph distribution between corals number CTW's 250 200 YELLOW 150 RED/WHITE 100 WHITE/TAN/PINK OTHER 50 23 21 19 17 15 13 11 9 7 5 3 1 0 coral head Graph 6: Y, RW, and WPT CTW color morph distribution between corals 35 30 yellow red/white white/pink/tan 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 # CTW 25 coral head Table 3: Chi-square analysis for the distribution of Y, RW, WPT & ‘Other’ CTW’s between coral heads Test Statistic Value dof Prob Pearson Chi-square 128.28 69 6.9x10-6 13 Table 4: Chi-square analysis for the distribution of Y, RW, and WPT CTW’s between coral heads Test Statistic Value dof Prob Pearson Chi-square 50.67 46 0.29 # of Free Standing CTW Color Morphs Graph 7: The number of single Y, RW, & WPT CTW morphs on 11 coral heads 5 4 3 2 1 0 0 2 4 6 8 CORAL HEAD 10 12 YSING RWSING WPTSING # of CTW's in Groups of Two of More Graph 8: The number of Y, RW, & WPT CTW morphs aggregated in groups of two or more, on 11 coral heads 5 4 3 2 1 0 0 MORETHAN2Y MORETHAN2RW MORETHAN2WP 2 4 6 8 CORAL HEAD 10 12 14 Table 5: Chi-square analysis for the age distribution of CTW’s grouped by color morph Test statistic Value Pearson Chi-square 6.588 df Prob 16.000 0.980 Graph 9: The number of CTW’s in different age groups catagorized by color morph y 1t 0 o1 11 to 20 21 to 30 31 to AGE 40 41 to 50 Count b/w Count b COLOR Count wpt Count r/w Count 10 9 8 7 6 5 4 3 2 1 10 9 8 7 6 5 4 3 2 1 10 9 8 7 6 5 4 3 2 1 10 9 8 7 6 5 4 3 2 1 10 9 8 7 6 5 4 3 2 1 15 85 1.2 84 1.0 83 0.8 RAIN TEMP Graph 10: The change in temperature, precipitation, wind speed (m/s) and waveheight (m) over time 82 0.6 81 0.4 80 0.2 79 0 5 10 15 20 0.0 0 25 5 10 DAY 15 20 25 15 20 25 DAY 9 2.8 8 2.4 WAVEHEIGHT 7 WIND 6 5 4 3 2 2.0 1.6 1.2 1 0 0 5 10 15 DAY 20 25 0.8 0 5 10 DAY