Zonation of Tegula Shi Jane Liaw ABSTRACT Two intertidal species of Tegula, T. brunnea and T. funebralis, display distinct vertical zonation. T. brunnea inhabit the lower intertidal to shallow subtidal zone, while T. funebralis are found in the upper-mid intertidal zone. Reciprocal transplants of each species to the habitat of the other revealed that T. brunnea returns to its typical height on the shore significantly more quickly than does T. funebralis. Transplanted T. brunnea and transplanted T. funebralis move significantly further and in a more consistent direction than do control animals placed back in their respective natural habitats. Although no statistical differences were observed between transplants done in high and low tides, a consistent trend of less movement by T. brunnea during low tides was observed. Microhabitat utilization during simulated low and high tides in the laboratory showed that T. brunnea remains submerged and on horizontal surfaces significantly more than T. funebralis, which tend to occupy vertical surfaces both above and below the waterline. Night and daytime behavior are similar for T. funebralis and differ for T. brunnea in the laboratory. Zonation of Tegula Shi Jane Liaw INTRODUCTION In order to understand the dynamics of a biological community, one must first understand the behaviors of its component organisms, including the reasons for their habitat choices. The rocky intertidal is an ideal community to study because of the ease of access to intertidal areas, the gradient of abiotic stresses to organisms caused by fluctuations in tidal height, and the abundance of many species (Connell, 1974). Two such species are Tegula funebralis and Tegula brunnea. Tegula funebralis is a gastropod abundant in the upper- to mid-intertidal zone between one and five feet above Mean Lower Low Water (Wara and Wright 1963, Abbott and Haderlie 1980) with a population center at about +3 feet. Tegula brunnea is found in the low intertidal to shallow subtidal zone, between 0 and 18 feet below MLLW (Abbott and Haderlie 1980, Watanabe 1984). These two common turban snails share many characteristics, including shell shape, size, diet and behavior. However, despite similar physiological characteristics, they are clearly vertically zoned. Because they are vertically zoned, abundant and move at a rate that is relatively easy to track, T. funebralis and T. brunnea are useful organisms in which to study habitat selection. While there has been substantial research on the zonation and migration behavior of T. funebralis (Wara and Wright 1963, Frank 1975, Markowitz 1980, Byers and Mitton 1981, Byers 1983, Fawcett 1984), little research has been done on T. brunnea zonation and migration. Thus, special interest in T. brunnea behavior motivated this study. In this study, a reciprocal transplant experiment and a laboratory experiment were conducted to examine the reasons for the ecological separation of the two species. Zonation of Tegula Shi Jane Liaw MATERIALS AND METHODS Vertical Zonation Two transects were sampled perpendicular to the shoreline at Hopkins Marine Station, Pacific Grove, CA, and one transect was sampled at Point Pinos, Pacific Grove, CA. Each transect was divided into four zones and all Tegula species in five 0.25m2 quadrats were counted within each zone. The positions of the quadrats along each transect were located using a list of random numbers. Heights at Point Pinos were measured relative to water level at the predicted time of low tide using a surveyor’s transit and stadia rod. Heights at Hopkins Marine Station were determined from a detailed topographic map of the intertidal zone. Laboratory Experiment Fifty snails of each species were placed in a 3 x 3m outdoor tank with running seawater. High and low tides were simulated during both day and night. To simulate high tides, a stand-pipe was placed in the drain which maintained a water depth of 10.1 inches in the tank. Observations were made every half-hour for at least an hour; during high tides the number of each species above the waterline, below the waterline and on the bottom of the tank were recorded. During low tides, the stand-pipe was removed and the water allowed to drain out of the tank. Counts were taken of snails on the walls and on the bottom surface of the tank every half- hour for at least an hour. Both day and nighttime sets of high and low tide experiments were replicated three times, with three different groups of snails. Analysis of Variance and Tukey Zonation of Tegula Shi Jane Liaw HSD tests were used to analyze differences in microhabitat utilization by T. funebralis and T. brunnea during high tides and low tides. Reciprocal Transplant Experiment, T. brunnea T. brunnea were transferred to the typical shore height of T. funebralis, up to heights between 2 and 4 feet above MLLW. Each set of high and low tide T. brunnea transplants were done at two locations, three times at one location and one time at the other. One of these sets of transplants was done during nighttime high and low tides. One hundred snails were scrubbed to remove algae from their shells and were then painted with flourescent enamel paints. During low tide transplants, snails were placed on air-exposed surfaces about 0.5 m away from the water edge. During high tide transplants, snails were placed under water at a depth of 0.3m to 0.5m. Transplants were checked 1.5 hours, 3 hours and approximately 24 hours after initiation of the experiment. Positions of snails found were recorded to the nearest meter from the release site; negative values were given to snails moving upshore and positive values were given to snails moving downshore. The average displacement of all snails found at each time point was calculated. Two controls were performed at high tide in which 100 T. brunnea were painted in a similar fashion to transplanted snails but were placed back to their typical height on the shore at about 4 feet below Mean Lower Low Water level. Measurements were taken after 24 hours, and average displacement was calculated as with transplanted T. brunnea. Two-tailed t-tests assuming unequal variances, with adjusted a-level 0.013, were performed between 24-hour displacement values of transplanted and control T. brunnea, between transplanted T. brunnea at high and low tides after 1.5 hours, Zonation of Tegula Shi Jane Liaw between transplanted T. brunnea at high and low tides after 3 hours and between transplanted T. funebralis and T. brunnea at high and low tides after 24 hours. This adjusted a-value was used because of lack of independence of the individual tests and was obtained using the Dunn-Sidak equation (Sokal and Rohlf 1995). A comparison between displacement values of transplanted T. brunnea at high and low tides after 24 hours was not made because after 24 hours, the snails had already experienced at least one high tide. A two-factor ANOVA test could not be used in place of the t-tests because data sets were incomplete; control T. brunnea were not counted at 1.5 hours and 3 hours. Reciprocal Transplant Experiment, T. funebralis T. funebralis were transplanted down to between 3 to 4 feet below MLLW, the highest point at which T. brunnea could be found in abundance. Two transplants were done at high tide, two transplants at low tide. Two sets of controls in which T. funebralis were placed back to their natural habitat at upper-mid intertidal were performed at high and low tides. Measurements of average displacement were calculated as for T. brunnea. A two-factor ANÖVA was performed with transplant-control as one factor and high tide-low tide as the second factor, in order to determine significance of differences within each factor and between each factor, and to determine significance of interactions between the two factors. Two-sample t-tests assuming unequal variances were also performed between T. funebralis and T. brunnea at high and low tides to determine significance of differences between displacements at these two tide heights. An ANÖVA test could Zonation of Tegula Shi Jane Liaw not be used in place of these t-tests because data sets were incomplete; T. brunnea controls were not performed at low tides. RESULTS Vertical Zonation Abundance of T. funebralis was greatest between 2 and 4 feet above MLLW and decreased with decreasing height on the shore. No T. brunnea were found at heights above MLLW (Fig. 1). The zone of maximum T. brunnea abundance was between 2 and 4 feet below MLLW. Densities of both species in the zones of overlap, at 0 to 4 feet below MLLW, were low (less than 25 T. funebralis, less than 10 T. brunnea per 0.25 m2 quadrat) compared to maximum T. funebralis densities (almost 75 T. funebralis per 0.25 m2 quadrat). Laboratory Experiment In all three replicates of experiments, T. brunnea tended to stay on the horizontal bottom of the tank significantly more than did T. funebralis (Tables 1 and 2). No T. brunnea were ever found above the waterline during high tides, while T. funebralis were regularly found above the waterline. When low tides were simulated by removal of the standing-pipe, T. brunnea visibly followed the receding waterline; often, in less than 10 minutes, all T. brunnea had moved down to the bottom of the tank. During nighttime low tides, more T. brunnea were found on the walls, with an average of 3% on the walls. For Zonation of Tegula Shi Jane Liaw daytime low tides, the average percentage of T. brunnea on the walls was less than 1%. T.brunnea were much more likely to climb and stay on the walls during high tides than during low tides. Twenty percent of T. brunnea were found on the walls during daytime high tides, below the waterline, while less than 1% were found on the walls during daytime low tides. T. funebralis did not avoid air exposure as much as T. brunnea, and did not follow the waterline as it receded during low tides. Numbers of T. funebralis on the wall were fairly consistent throughout day and nighttime, high- and low-tide transplants. T. funebralis on walls and on the bottom surface did not exhibit any movement when water was drained for low tides. Reciprocal Transplant Experiment, T. brunnea T. brunnea transplanted to heights between 2 and 4 feet above MLLW migrated significantly (t = 9.55, p««0.01) more after 24 hours than control T. brunnea placed at their typical shore height (Fig. 2). Transplants done during high and low tides (Fig. 3) did not display significant differences (t = 3.15, p » 0.05) in migration rate after 3 hours. Transplants done during high and low tides after 1.5 hours also did not display significant differences and trends were similar to that of transplants after 3 hours and thus, results were not graphed. Reciprocal Transplant Experiment, T. funebralis T. funebralis transplanted to heights between 3 and 4 feet below MLLW migrated significantly more (ANOVA, p « 0.05) than control T. funebralis placed at their typical shore height (Fig. 4). Transplants done during high and low tides Zonation of Tegula Shi Jane Liaw (Table 3) did not display any significant differences (ANOVA, p » 0.05) in migration rate. Comparison of T. funebralis and T. brunnea movements T. brunnea and T. funebralis migrated to their typical heights on the shore at significantly different rates both at high tides (t =-10.78, p ««0.001) and at low tides (t = -8.76, p «20.001). T. brunnea moved much faster downshore after both high tide and low tide transplants (Fig. 5). DISCUSSION From this study, it appears that Tegula funebralis tolerate exposure to air more than Tegula brunnea. T. brunnea generally avoids movement when exposed, while T. funebralis exhibits a climbing behavior and prefers vertical faces to horizontal surfaces, even when exposed. Although differences in displacement of transplanted T. brunnea at high and low tides were not significant,T. brunnea moved much faster downshore when they were transplanted under water during high tides than when they were transplanted at low tides and exposed to air. In three of the low-tide transplants, no snails moved for the first three hours, and in the fourth and last transplant, only three snails moved less than 1 meter away from the release site. Transplanted T. brunnea moved not only further than controls, but also in a more consistent direction. The majority of transplanted T. brunnea moved downshore, whereas movement of control animals placed back into their natural low shore habitat was more scattered in both directions. Zonation of Tegula Shi Jane Liaw Transplanted T. funebralis could not be counted consistently at 1.5 hours and 3 hours because of rough water conditions and so a comparison of high and low tide displacements at 3 hours could not be made. However, field observations indicate that T. funebralis are more mobile than T. brunnea when exposed to air and tend to move until they find a suitable area to cluster with other T. funebralis. In the laboratory experiment, when water was drained out for the low tide, T. brunnea followed the waterline to the bottom of the tank within an average of approximately five minutes. In contrast, T. funebralis did not move from their positions in clusters above the waterline. While T. brunnea preferred the bottom of the tank during both day and night low tides, at least two and up to six T. brunnea stayed on the walls during low tide at night. In contrast, only one T. brunnea was ever on the wall at once during daytime. In contrast, there were no significant changes in behavior of T. funebralis during different times and tides. The migration behavior of the two species indicates that the upper limit of the T. brunnea zone may be caused by an inability to tolerate heat and desiccation, while the lower limit of the T. funebralis zone may be caused by an innate climbing behavior. T. brunnea were never found above the waterline in the laboratory experiment, they followed the waterline down at low tide and at lower temperatures during the night, they were slightly more likely to stay on the walls during low tide. In addition, T. brunnea did not move during low-tide transplants and instead retracted into their shells until the water level rose to cover them. A previous study also reveals that a combination of heat stress and water loss probably determines how long T. brunnea can survive out of water (Rocca 1995). Rocca (1995) found that when subjected to similar heat and desiccation treatments, T. funebralis had a much higher survival rate than T. brunnea. It has been further 10 Zonation of Tegula Shi Jane Liaw demonstrated that, in comparison with organisms living lower, higher intertidal animals tend to have higher desiccation tolerances and lower desiccation rates (Wolcott 1973). Climbing behavior of T. funebralis could be responsible for the lower limit of their distributions. Both T. funebralis in the field and in the laboratory exhibited this behavior. The reason for this is unknown, but it is possible that it was once an attempt to avoid predators (Paine 1969) that has since evolved into behavior. T. funebralis may have evolved this behavior while T. brunnea did not because a major predator of the two species, Cancer antenarius, finds it easier to crack the shells of T. funebralis than T. brunnea (Abbott and Haderlie 1980). Thus, T. funebralis have a disadvantage in encounters with the predator Cancer. While Rocca (1995) showed that physiological tolerance rather than behavior allows T. funebralis to live in the high intertidal, this study indicates that both factors play a part in their habitat selection. T. funebralis are better able to tolerate heat and desiccation stress than T. brunnea and they have also developed a climbing behavior that may allow them to escape predators which do not feed above the waterline, such as Cancer antenarius and Pisaster ochraceus. Research has been done that demonstrates T. funebralis exhibit climbing behavior in the presence of predators (Fawcett 1984, Markowitz 1980, Paine 1969). In the laboratory component of this study, T. funebralis climbing behavior is shown to occur even when no predators are present. Öther factors may also be responsible for T. funebralis and T. brunnea zonation, including larval dispersal. Larval recruitment is an important cause of the spatial segregation of the three subtidal Tegula species (Watanabe 1984) and it is known that T. funebralis larvae settle high in the intertidal zone (Paine 1969) and T. 11 Zonation of Tegula Shi Jane Liaw brunnea larvae settle in the low intertidal to shallow subtidal zone (Watanabe 1984). Larval recruitment may therefore play a part in the segregation of these two species as well. However, this theory cannot fully explain why the snails would migrate towards their typical shore habitat after being transplanted. Microhabitat preferences may also be partially responsible for the zonation of T. funebralis and T. brunnea. Byers and Mitton (1981) found that after translocation T. funebralis return to their natural habitats through a recognition of ecological characteristics of the habitats. T. brunnea are found most often in areas of dense algal cover (Watanabe 1984) while T. funebralis avoids dense algal cover (Wara and Wright 1963). It is thus possible that in the reciprocal transplants of this study, T. funebralis and T. brunnea were migrating to areas of preferred microhabitat. Even so, other factors must be considered in combination, for microhabitat preference alone cannot explain how snails are able to discern their location when transplanted, and move in the correct direction towards their natural habitats. Interspecific competition does not appear to influence zonation patterns between T. funebralis and T. brunnea, since intraspecific densities of T. funebralis are much greater (about 75 T. funebralis per 0.25m2 quadrat) than interspecific densities where the two species overlap (less than 25 snails per 0.25m2 quadrat). Furthermore, Watanabe (1984) has shown that interspecific competition is not higher than intraspecific competition between T. brunnea and the two other subtidal Tegula species, T. montereyi and T. pulligo. This may also the case between T. brunnea and T. funebralis. While Wara and Wright (1963) demonstrated that T. funebralis movement was light-dependent, with snails moving up to rock tops during night high tides and not day high tides, this study found no significant differences between the 12 Zonation of Tegula Shi Jane Liaw number of snails at higher latitude in the tank during day and night. However, further investigation would be of value. The differences in displacements of transplanted T. funebralis and T. brunnea, with T. brunnea moving significantly longer distances than T. funebralis, show that the factors limiting T. brunnea are probably more direct than those limiting T. funebralis. This is in keeping with previous studies (Dayton 1971, Paine 1974) that show that upper limits of intertidal organism range tend to be caused by abiotic factors, while lower limits tend to be caused by biotic interactions. It is also in keeping with the theory that heat and desiccation may be the cause of T. brunnea zonation and an evolved climbing behavior may be the cause of T. funebralis zonation. ACKNOWLEDGEMENTS I would like to thank my advisor, Jim Watanabe, for his guidance, dedication and boundless knowledge of snails and other intertidal creatures. I would also like to thank my "snorkel buddies", especially Josh Rapport, without whom I could never have found and counted that many tiny fluorescent snails. Zonation of Tegula Shi Jane Liaw LITERATURE CITED Abbott, D. P. and E. C. Haderlie. 1980. Prosobranchia: Marine snails. Pages 252-254. R. H. Morris, D. D. Abbott and E. C. Haderlie, ed. Intertidal Invertebrates of California. Stanford University Press, Stanford, CA. Byers, B. A. 1982. Enzyme polymorphism associated with habitat choice in the intertidal snail Tegula funebralis. Behavior Genetics 13: 65-75. Byers and Mitton, 1981. Habitat choice in the intertidal snail Tegula funebralis. Marine Biology 65: 149-154. Connell, J. H. Effects of competition, predation by Thais labillus. Ecological Monographs 31 (1): 83-104. Dayton, P. K. 1971. Competition, disturbance, and community organization: the provision and subsequent utilization of space in a rocky intertidal community. Ecological Monographs 41: 351-389. Fawcett, M. H. 1984. Local and latitudinal variation in predation on an herbivorous Marine Snail. Ecology 65 (4): 1214-1230. Frank, P. W. 1975. Latitudinal Variation in the life history features of the black turban snail Tegula funebralis (Prosobranchia: Trochidae). Marine Biology 31: 181-192. Markowitz, D. V. 1980. Predator influence on shore-level size gradients in Tegula funebralis. Journal of Experimental Marine Biology and Ecology 45: 1-13. Paine, R. T. 1969. The Pisaster-Tegula interaction: prey patches, predator food preference, and intertidal community structure. Ecology 50 (6): 950-961. Paine, R. T. 1974. Intertidal community structure: experimental studies on the relationship between a dominant competitor and its principal predator. Oecologica 15: 93-120. 14 Zonation of Tegula Shi Jane Liaw Underwood, A. J. 1979. The ecology of intertidal gastropods. Advanced Marine Biology 16: 111-210. Wara, W. M. and B. B. Wright. 1963. The distribution and movement of Tegula funebralis in the intertidal region of Monterey Bay, California. Veliger 6 supplement: 30-37. Watanabe, J. M. 1984. The influence of recruitment, competition, and benthic predation on spatial distributions of three species of kelp forest gastropods (Trochidae: Tegula). Ecology 65 (3): 920-936. Wolcott, T. G. 1973. Physiological ecology and intertidal zonation in limpets (Acmaea): a critical look at "limiting factors. Biological Bulletin 145: 389-422. Zonation of Tegula Shi Jane Liaw FIGURE LEGENDS Vertical Distribution of Tegula funebralis and T. brunnea at Hopkins Figure 1. Marine Station and Point Pinos. Error bars show 95% confidence intervals. Figure 2. Average Displacements of Transplanted and Control T. brunnea put out during high tide, at 24 hours (t = 9.55, p «« 0.001). Error bars show 95% confidence intervals. Figure 3. Average Displacements of Transplanted T. brunnea at high and low tides after 3 hours (t = 3.15, p» 0.05). Error bars show 95% confidence intervals. Figure 4. Average Displacements of Transplanted and Control T. funebralis (ANÖVA, p « 0.05) after 24 hours. Error bars show 95% confidence intervals. Average Displacements of T. funebralis and T. brunnea at high tide (t = Figure 5. -10.78,p «« 0.001) and low tide (t =-8.76, p «« 0.001) after 24 hours. Error bars show 95% confidence intervals. Zonation of Tegula Shi Jane Liaw Figure 1. 204 . to2. E T. funebralis E T. brunnea 210 0 410 2 2 50 75 25 100 Average number of snails per 0.25m22 quadrat Zonation of Tegula Shi Jane Liaw transplant control Figure 2. S T. brunnea Zonation of Tegula Figure 3. 1.5 0.5 O- High Low tide height when snails released Shi Jane Liaw ET. brunnea Zonation of Tegula Figure 4. 0.5 . . -0.5- high low tide height when snails released Shi Jane Liaw E transplants controls Zonation of Tegula Figure 5. . high low tide height when snails released Shi Jane Liaw E T. funebralis ET. brunnea 5 + 1+ 20 u 55 + 4. Shi Jane Liaw Zonation of Tegula P-value F-Ratio degrees of Mean- Sum of Source of freedom Square Squares Variation 24.49 5.72 5.72 Species 41.69 9.73 Location 9.73 148.13 34.56 34.56 Interaction 0.23 20 467 Error TWO-FACTOR ANOVA WITH SPECIES (T. FUNEBRALIS-T. TABLE 2a. AS ONE FACTOR AND LOCATION (ON WALL-ON BOTTOM) AS BRUNNEA OTHER FACTOR, FOR SIMULATED LOW TIDE IN TANK EXPERIMENT. 1.000 1.000 1.000 1.000 0.714 0.003 4 TABLE 2b. TUKEY HSD MULTIPLE COMPARISONS. MATRIX OF PAIRWISE COMPARISON PROBABILITIES. 1—T. brunnea on bottom of tank. 2—-T. brunnea on wall of tank. 3—-T. funebralis on bottom of tank. 4—T. funebralis on wall of tank. Shi Jane Liaw Zonation of Tegula P-Value Mean- F-Ratio degrees of Sum of Source of Squares freedom Square Variation 25.38 Species 6.81 6.81 57.03 30.59 15.30 Location 15.04 56.06 Interaction 30.07 0.268 Error 8.05 30 — TABLE 2c. TWO-FACTOR ANOVA WITH SPECIES (T. FUNEBRALIS-T. BRUNNEA) AS ONE FACTOR AND LOCATION (ABOVE THE WATERLINE¬ BELOW THE WATERLINE-ON BOTTOM) AS OTHER FACTOR, FOR SIMULATED HIGH TIDE IN TANK EXPERIMENT. 1.000 1.000 0.007 1.000 1.000 1.000 0.017 0.658 1.000 0.458 0.371 0.997 0.369 1.000 0.005 TABLE 2d. TUKEY HSD MULTIPLE COMPARISONS. MATRIX OF PAIRWISE COMPARISON PROBABILITIES. 1—T. brunnea above waterline. 2—T. brunnea below waterline. 3—T. brunnea on bottom of tank. 4—T. funebralis above waterline. 5—T. funebralis below waterline. 6—T. funebralis on bottom of tank. Zonation of Tegula Shi Jane Liaw F-critical F-value P-value Mean- Source of degrees of SS value Variation Square freedom 0.199 2.36 7.71 0.198 High tide¬ 0.198 Low tide 119 7.71 1.006 0.026 1.006 Transplant -Control 0.838 7.71 0.004 0.004 004 Interaction Within 0.083 0.336 Total 1545 TABLE 3. TWO-FACTOR ANOVA WITH HIGH TIDE-LOW TIDE AS ONE FACTOR AND TRANSPLANT-CONTROL AS THE OTHER FACTOR, FOR TEGULA FUNEBRALIS AT 24 HOURS.