DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES Degree project for Bachelor of Science with a major in biology BIO603 Biologi: Examensarbete 30 hp First cycle Semester/year: Spring 2017 Supervisor: Kerstin Johannesson, Department of Marine Sciences Examiner: Susanna Eriksson, Department of Biological and Environmental Sciences THE EFFECT OF SIZE DIFFERENCE AND ECOTYPE ON THE RISK OF LITTORINA SAXATILIS GETTING SWEPT AWAY BY WAVES DURING MATING Elin Nilsson
Transcript
DEPARTMENT OF BIOLOGICAL AND
ENVIRONMENTAL SCIENCES
Degree project for Bachelor of Science with a major in biology BIO603 Biologi: Examensarbete 30 hp First cycle Semester/year: Spring 2017 Supervisor: Kerstin Johannesson, Department of Marine Sciences Examiner: Susanna Eriksson, Department of Biological and Environmental Sciences
THE EFFECT OF SIZE DIFFERENCE AND ECOTYPE ON THE RISK OF LITTORINA SAXATILIS GETTING SWEPT AWAY BY WAVES DURING MATING
Elin Nilsson
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Table of Contents Abstract ............................................................................................................................................ 2
Abstract The direct developing periwinkle Littorina saxatilis has the highest mounting probability when the female is roughly double the size of the male. A previous bachelor’s thesis showed that mounted females are more likely to be swept away by running water than other females, and this likelihood was larger in a group of females with males larger than themselves, compared to females mated with males of similar or smaller sizes. This thesis raised the same issue but compared female/male pairs of many different relative sizes and of both exposed wave ecotype and the sheltered crab ecotype of Littorina saxatilis where the wave ecotype is under stronger wave stress in the wild than is the crab ecotype. Females were placed in a high-speed aquarium with shells of males glued to their back in mounting position, and the water speed at which they fell off were noted. Results included 160 female snails from Saltö in Kosterhavet, 88 of the crab ecotype and 72 of the wave ecotype. There was a significant correlation between the water speed at which the females fell off and their relative size to the male. There was a greater difference in mean between when the female was larger and the male was larger for the wave ecotype, but it was not significant. In conclusion, both size and ecotype seem to affect the risk of being swept away, but it’s only significant for size.
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Introduction Littorina saxatilis (Olivi) is an intertidal rough periwinkle common in the North Atlantic which inhabits rocky and boulder shores (Reid 1996). The snail has ecotypes adapted to different habitats with inherited differences in size and shape (Janson 1982; Johannesson et al. 1993). It has been suggested that these inherited differences are part of an ongoing ecological speciation; there is strong evidence that the divergence of the ecotypes is happening in the presence of gene flow (Butlin et al. 2014), and that the ecotypes are partially reproductively isolated.
There are many speculations as to what might be the source of the differences between the ecotypes. The snail is ovoviviparous, lacks pelagic larval dispersal, and has a low adult dispersal (1-4 m/month), leading to low gene flow between different populations (Janson 1983). Another factor might be that despite the small geographical difference, the ecotypes have very different habitats and face different threats. On Saltö, a Swedish island in Kosterhavet, there are two ecotypes of L. saxatilis: the wave ecotype, and the crab ecotype, which occur in repeated intervals of crab ecotype and wave ecotype, with hybrid zones in between (Janson 1982). The crab ecotype is found below or slightly above sea level in boulder bays (Janson 1983), where it’s protected from waves but is exposed to crabs and moving rocks, and is therefore benefited from having a small aperture (opening), a thick shell, and a low spire (Janson 1982). Experiments show big, thick shells with a low spire withstand crab attacks better than small, thin, high-spired shells (Johannesson 1986). The wave ecotype, on the other hand, is usually found on cliffs (Janson 1983), high up in the splash zone where crabs can’t reach it, but where it is exposed to waves (Janson 1982). It is smaller, but with a relatively larger aperture (allowing for a larger area connected to the rock), and a thin shell (Janson 1982). The females of L. saxatilis are larger than the males (Reid 1996), and although there has been some contradicting studies whether or not both males of both ecotypes prefer larger females (Erlandsson and Rolán-Alvarez 1998), recent, extensive experiments in lab suggest that both ecotypes do (unpublished data/data in preparation, see Appendix 1 for a graph showing the relationship between relative size and mounting probability). In a study on Littoraria ardouiniana, Ng and Williams (2014) showed similar results; while small males were unselective, large males preferred to follow mucus trails laid by large females, and both sizes copulated for a longer time with larger females. The appearance of small males being unselective might, however, be an effect of the males preferring females that are either the same size or larger than them (Ng and Williams 2014). Another example comes from Littorina littorea, where males from some (but not all) populations prefer to mate with large females (Erlandsson and Johannesson 1994). There are a number of theories for why this preference for large females exist, the most obvious one being that female fecundity increases with female size in littorinid snails (Janson 1985). Another theory is that when the male is equal to or larger than the female, the risk that they will be swept away by waves during mating increases. Saur observed that small females sometimes lose their grip on the rock surface when large males try to mate them (pers. comm., cited from Erlandsson and Johannesson 1994), and snails that have been swept away in this manner rarely make it back (K. Johannesson, pers. observation). Results from a bachelor’s thesis show that for the crab ecotype, when the male was equal to the female in size, or larger than her, the risk for the female to lose her grip was higher than if not mounted. When the female was almost double the size of the male, there was no difference between mounted and unmounted females (Thorén 2016). This effect of the relative size on the fall-off risk should be even more important to the wave ecotype, given that they are more exposed to waves than the crab ecotype, and the size difference between the sexes seems to be greater within the wave ecotype than within the crab ecotype (Erlandsson and Rolán-Alvarez 1998).
This thesis compared the wave and crab ecotypes of Littorina saxatilis and the effect of the relative size between the female and male during mounting when impacted by the force of flowing water. Essentially, testing the female’s ability to resist being swept away at different
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water speeds. Since the wave ecotype is adapted to waves and has a relatively larger foot, it might be that it is less sensitive to a lower or negative relative size. Another hypothesis is that, because the size difference between the sexes is greater within the wave ecotype, it might be more sensitive to a lower or negative relative size.
Material and Method Test organism Both ecotypes (wave and crab) of the species Littorina saxatilis were collected from sites on the island of Saltö (Fig. 1), during May-June. In total, measurements were obtained for 88 of the crab ecotype and 72 of the wave ecotype. Female sizes ranged between 5-12 mm aperture diameter in the crab ecotype, and 3-10 mm aperture diameter in the wave ecotype.
The snails were sexed based on the presence or absence of a penis. The snails that were glued onto the females were boiled and the soft parts removed. Since there is no difference in shell shape between females and males, shells of females were also used as "male" shells. The ”males” were glued onto the females in a typical mating position (see Fig. 2).
Snails were stored at a temperature of 5°C for a maximum of Y days before used in the experiment.
Experimental design The longest distance off the opening was measured (see Fig. 3), which was used to calculate the relative size of the female to the male, using the equation below.
Relative size = !"!!!"!!"!!!"!
, where ALF is AL for the female, and ALM is AL for the male (see Fig.
3 for what AL means).
Figure 1: Showing Saltö, outside Tjärnö in Strömstad, Sweden. The localities the snails were sampled are marked; the wave ecotype from point A and the crab ecotype from point B. The maps are screenshots from google maps.
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Figure 2: The typical mating position of Littorina saxatilis. Photo taken by Patrick Larsson.
Females with glued male shells were placed up to five at a time in the experimental tube (see fig 4) of the high-speed aquarium. When they had attached to the wall of the tube the snails were allowed to get accustomed for ten minutes, with the water completely still. After ten minutes, the valve was opened. There were in total five positions with different water speeds. The water was allowed to flow until the water in the tank ran out, after which it was noted which snails had fallen off, and the remaining snails were given two minutes to rest. If a snail did not fall off at the highest speed of the first tube it was removed from this tube and later (during the same day) tested in a smaller tube with even higher water speed. Thus, in total, ten different water speeds were
tested, ranging from 0,75-3,6 m/s. Based on previously recorded data, the valve positions were converted to speed (m/s).
Figure 3: Shell dimensions, where AL is the longest distance of the opening. (Janson 1982)
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Statistics Graphs and all statistical calculations were done using SPSS. Mean fall-off water speeds were compared with Mann-Whitney U when there were two categories on the x-axis, and with Kruskal-Wallis and a Dunn-Sidak corrected Mann-Whitney U when there were four categories. Correlation coefficients and significance were obtained with Spearman Rank Correlation.
Figure 4: The high-speed aquarium used during the experiments. The raised tube in the aquarium was were the snails were placed. Water flowed from the tank in the background, up through the tubes connecting to it to a raised tank to the right of the picture (not visible). When the valve on the left side of the picture was opened, the water flowed through the tube, back into the tank. Photo taken by author.
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Results For both crab and wave ecotype there was a significant difference between the mean water speed at which the females fell off when comparing pairs with larger females to pairs with larger males (crab: U38,50 = 575,5 P = 0,001, wave: U58,14 = 182,5 P = 0,001) and the difference seemed larger for the wave ecotype than for the crab ecotype (Figs 5 and 6). When comparing the relative sizes
between the two ecotypes (i.e. larger males for the crab ecotype vs larger males for the wave ecotype) there were no significant differences between either relative size (larger male: U14,50 =
262 P = 0,140, larger female: U58,38 = 936 P = 0,204).
When the relative sizes are divided into four groups, the mean values get higher from the relative sizes -0,5 to 0,25 (see table 2 for exact values), and at the relative sizes 0,25 – 0,5 it gets lower than the previous size group (see Fig. 7). However, the error bars are quite big, especially for the last size group, and there’s no significant difference between it and any of the other groups. For the wave ecotype the mean values continue getting higher (see Fig. 8). There were significant differences between the groups in both ecotypes (crab: K3 = 10,996 P = 0,012, wave: K3 = 11,857
Figure 5: The effect of relative size (here divided into bigger female/male) on the water speed (m/s) that caused the female to fall off (mean fall-off water speed (m/s) ± 1 SE) for the crab ecotype. Relative size refers to which snail was bigger, the mounting male or the female. See Table 1 for N, exact means, and standard deviations.
Crab ecotype Wave ecotype
Figure 6: The effect of relative size (here divided into bigger female/male) on the water speed (m/s) that caused the female to fall off (mean fall-off water speed (m/s) ± 1 SE) for the wave ecotype. Relative size refers to which snail was bigger, the mounting male or the female. See Table 1 for N, exact means, and standard deviations.
Figure 7: The effect of relative size on the waters peed (m/s) that caused the female to fall off (mean fall-off water speed (m/s) ± 1 SE) for the wave ecotype. Relative size refers to which snail was bigger; if the value is below than zero the male was bigger, if the value is above than zero the female was bigger. See Table 2 for N, exact means, and standard deviations.
Figure 8: The effect of relative size on the water speed (m/s) that caused the female to fall off (mean fall-off water speed (m/s) ± 1 SE). Relative size refers to which snail was bigger; if the value is below than zero the male was bigger, if the value is above than zero the female was bigger. Se Table 2 for N, exact means, and standard deviations.
Crab ecotype Wave ecotype
8 Figure 12: The effect of the relative size (where a relative size about zero means the female was bigger than the mounting male, and a relative size below zero means the male was bigger) on the opening width (mm). Crab: y = 7,42 + 7,77*x. R2 = 0,824. Wave = y = 3,9 + 5,41*x. R2 =0,589.
Figure 10: The distribution of the relative size divided into the ecotypes.
P = 0,008). With the Dunn-Sidak corrected Mann-Whitney U the difference is significant at P ≤ 0,008512, and for the crab ecotype there were significant differences between group one and three (U12,27 = 77 P = 0,008) and two and three (U38,27 = 298 P = 0,003), but not any other groups. For the wave ecotype, there’s only a significance difference between group two and four (U41,17 = 289 P = 0,3). The line for the effect of relative size on the water speed the females fell off at is more pronounced for the wave ecotype, with a steeper curve for the wave ecotype (see Fig. 9), but R2 is rather low for both ecotypes. There was significant correlation between relative size and fall-off water speed for both ecotypes (crab: p86 = 0,367 p < 0,001, wave: p70 = 0,355 p = 0,002). The variance of the relative size was similar between the ecotypes, but the average was higher for the wave ecotype than for the crab ecotype (see Fig. 10).
The opening width, here used as a measure of the female’s size, had no significant effect on the fall-off water speed. (see Fig. 11). There is a relationship between relative size and opening width, with larger relative sizes being correlated to larger opening widths (see Fig. 12).
Figure 11: The effect of the opening with (mm) on the water speed that caused the snails to fall off. Wave ecotype: y = 4,63 + 2,9*x – 0,38*x^2 + 0,02*x^3. R2 = 0,239. Crab ecotype: y = 1,14 + 0,03*x + 6,85E-3*x^2 – 1,15E-4*x^3. R2 = 0,105.
Figure 9: Effect of the relative size on the water speed (m/s) that caused the female to fall off for both the crab ecotype and wave ecotype, and a curve fits for both. Relative size refers to which snail was bigger; a relative size above zero means the female was bigger than the mounting male. Crab ecotype: y = 1,74 + 1,27*x – 0,59*x^2 – 1,93*x^3. R2 = 0,110. Wave ecotype: y = 1,74 + 2,6*x – 0,08*x^2 – 4,36*x^3.
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Discussion For both ecotypes, the relative size of the female to the male seems to affect the risk that the female will lose her grip, leading to the couple falling off during mating, and the relationship seems to be steeper for the wave ecotype. One explanation for this is that the wave ecotype had higher relative size values (meaning that the size difference between the male and the female was greater, in the direction that the females were larger), which means that they would make the mean in Fig. 10 higher. However, since the regression line for the wave ecotype is more pronounced in Fig. 9 there still seems to be a difference between the ecotypes, despite what effect the uneven distribution in relative sizes might have on the means of the fall-off water speed. The wave ecotype also had a more consistent relationship between relative size and mean fall-off water speed than the crab ecotype; in Fig. 7, the last group of relative sizes has a lower mean than the previous one does (however, since the error bars were quite wide, this needs further testing). There are multiple explanations for this. The snail might have gotten so big that the relative size had less effect than the actual size of the snail. Another explanation is a behavioural one: the crab ecotype is generally shyer than the wave ecotype; they take longer to come out of their shell, and are more easily frightened. This shyness seemed to strengthen with size, so there could be that the greater relative sizes fell off easier because the females were larger, and therefore more easily frightened. However, there doesn’t seem to be relationship between opening width and fall-off water speed, but that might be because opening width is not a reliable measurement for size. It would have been better to the from the top of the spire to the bottom of the opening (W in Fig. 3). The previous bachelor’s thesis looked at how the relative size affects the fall-off risk for the crab ecotype, and although it only looked at three relative sizes (0,2, 0, and -0,18), the results were similar; the female’s fell off at higher speeds when the relative size was positive (Thorén 2016). This corroborates the relative sizes up to 0,2, at least, although since the relative sizes did not go above 0,2 it’s not possible to see if there would have been the same sudden dip in fall-off water speed.
When comparing the regression lines in Fig. 9 and the graph in appendix 1, the lines have the same general shape, but where the lowest and highest values are on the x-axis don’t quite seem to match up; in Fig. 9, the wave ecotype has a highest value at relative size about 0,42 and a lowest at roughly -0,40, the crab ecotype has a highest at roughly 0,35 and a lowest somewhere lower than -0,40. In the graph in appendix one, mounting probability is highest at a relative size of roughly 0,25, and lowest at a lowest somewhere lower than -0,5. This is to be taken with a grain of salt, however, since the R2 is so low for the fall-off graphs. The highest and lowest values are on the same side of zero, and in a rough vicinity of each other, so there might be a relationship. It would be easier to examine with the actual data of the mounting probability, which would certainly be interesting to examine. If the male is in any way conscious off the risk he faces when he copulates with a smaller female, this could add another factor for why the males prefer larger females (on top of larger females having a higher fecundity). Even if he isn’t conscious of the risk, natural selection would act against males that copulate with females smaller than themselves, and these males (and the females) would get swept away. Because of their low dispersal, it’s unlikely that they would get back to their original population (it’s likely that they would get eaten by fishes and crabs in deeper water). This in itself could also be another factor for the size difference in males and females: large males (and to some degree, small females) are disadvantaged, because they have more difficulties finding a female that is larger than them, and therefore safe to copulate with. The wave ecotype has a greater size difference between the sexes than the crab ecotype (Erlandsson and Rolán-Alvarez 1998), and although this might be because of different reproductive strategies (on Saltö, the crab ecotype has more absolute embryos, but when plotted
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against the size and weight of the female, the wave ecotype has relatively more (Janson 1985)), it might also be because the relationship between relative size and the fall-off risk is steeper for the wave ecotype. Why they seem to be more sensitive to negative relative sizes is difficult to answer from these results (and other studies); since they have a relatively larger foot, and are adapted to being exposed to waves, it feels logical that they should be less sensitive to size differences. One theory is that the greater size difference is because of the reproductive strategies, and that because the wave ecotype’s reproductive strategy leads to them having a greater size difference, they are adapted to having a greater positive size difference when mating. If the crab ecotype has a lesser size difference because of its reproductive strategy, they are adapted to this smaller size difference, and so are less sensitive to a negative size difference. This is, of course, very hypothetical, and even if further experiments get more definite results, it’s not possible to say that the different reproductive strategies have led to the wave ecotype being more sensitive to negative size differences. The relative size of the female and male affects both the wave and crab ecotype, but it seems like it affects the wave ecotype more.
Acknowledgements Thank you to Kerstin Johannesson, who was an amazing supervisor, and also to everyone at the Lóven Centre Tjärnö – especially to the people in the workshop who helped fix the high-speed aquarium when it suddenly decided to not work, and to Samuel Perini.
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Ng, T. P. T., and G. A. Williams. 2014. Size-Dependent Male Mate Preference and its Association with Size-Assortative Mating in a Mangrove Snail, Littoraria ardouiniana. Ethology 120:995–1002.
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Reid, D. G. 1996. Systematics and evolution of Littorina. London: The Ray Society. 463 p. Thorén, A. 2016. Påverkan av förändrat vattenmotstånd för Littorina saxatilis honor under
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Appendix 1
Figure 13: The effect of the relative size (calculated according to formula 1) between the female and the male on the probability that the male will mount the female. (R.K Butlin, pers. commun)
