Heredity 64 (1990) 169—175 The Genetical Society of Great Britain Received 4 August 1989
Inbreedingdepression and self-fertilization in Lymnaeaperegra (Gastropoda: Pulmonata)
P. Jarne and Université Montpellier II, B. Delay Institut des Sciences de l'Evolution, Laboratoire "génétique et environnement", Place E. Bataillon, 34000 Montpellier, France.
The effects of self-fertilization and cross-fertilization on several fitness traits were examined in the freshwater hermaphrodite snail Lymnaea peregra. Laboratory strains were established from Lake Geneva populations. Comparisons of F2 snails and their offspring showed that there are no differences in hatching time, nor in the size of young snails monitored over one month. But there was a significant difference, when the distribution of the capsule weight against the number of eggs was compared, although the effects of this on fitness are probably small. There was also a significant difference for egg production and juvenile viability over one month; the selfing snails are 94 per cent less fit for these two traits than the outcrossing.
INTRODUCTION self-fertilization rate gradually increases (Duncan, 1975). L. peregra preferentially cross-fertilizes Self-fertilizationand inbreeding depression have whenever allosperm is available (Boycott et a!., been widely studied, from both a theoretical and 1930). Thus, selfed products must be obtained from an empirical point of view (Lloyd, 1979; Lande individuals which have been isolated when and Schemske, 1985; Charlesworth and Charles- immature. worth, 1987; Holsinger, 1988; Schemske and Lande, 1985; Stevens and Bougourd, 1988). Most studies are limited to plants. However, mating sys- MATERIALSAND METHODS tems are as diverse in molluscs as in plants. All pulmonates are hermaphroditic (Duncan, 1975) Parentsnails were collected on the littoral of Lake and numerous Basommatophora species self- Geneva in June 1987. The animals were kept in fertilize. Comparative data on the fitness of the laboratory in 3-litre glass tanks. Egg capsules individuals in cross-fertilization and self-fertiliz- were collected every 3 days. The hatchlings (Fl ation are important for understanding the evol- generation) were reared from these. The capsules ution of natural populations. However, few studies of the Fl generation were collected and those have been undertaken in molluscs (Selander and designed for self-fertilization were incubated Kaufman, 1973; Selander et a!., 1975). We present individually in 150 ml plastic boxes. After hatch- data on such a comparison at different stages of ing, young F2 snails were isolated in similar boxes. the life cycle in the Basommatophoran, Lymnaea Capsules designed for the cross-fertilization study peregra. The ratio of self-fertilization vs. cross- were incubated in 3-litre glass tanks. Thirty young fertilization results measures the phenotypic effects snails at the most were reared per tank. The data of self-fertilization on some components of fitness. on inbreeding depression were obtained on the F2 L. peregra is a freshwater snail from Europe generation and their offspring. Throughout the and West-Asia (Hubendick, 1951). It is a func- study, all snails were maintained at 20°C with an tional self-fertile hermaphrodite (Diver et a!., artificial photoperiod of 16L/8D. They were fed 1925). Boycott et a!. (1930) showed that foreign with lettuce and water was changed about every 3 sperm is stored and remains viable for several days. months after copulation. It is used for cross- In Experiment 1, the oviposition rate and the fertilization. As it becomes exhausted or dies, the capsule weight of F2 snails are compared. The 170 P. JARNE AND B. DELAY study was conducted on 12 selfing snails and four The size at birth of three haphazardly groups of 10 outcrossing snails. The capsules (F3 individuals chosen per capsule was generation) were collected over 40 days. As the measured. When less than three individuals number of individuals and oviposition rate was were available, particularly for self-fertiliz- higher in cross-fertilization, only 200 randomly ation, only one or two individuals were chosen capsules were used in the analysis. The measured. T0 is the date corresponding to number of eggs per capsule was counted and the the mean hatching time. capsules were desiccated at 70°C for 24 hours (2) The percentages of survivors and S2c before they were weighed to an accuracy of andsize of three of them at respectively T1 1/1000 mg. and T2. The number of eggs per capsule and the weight of the egg capsules were log-transformed to stabil- Sic° (N1/N0) * 100 ize the variance. Mean egg numbers were com- pared between outcrossed and selfed capsules with S2c= (N2/N1) * 100 a t-test. The oviposition rate was calculated as the number of eggs and the number of capsules per where N1 and N2 are the number of sur- snail and per day. For all data and data of each vivors. T1 and T2 are respectively on average 14 and 30 days after T0. mating system, the capsule weight was plotted against the number of eggs per capsule. The linear Before the statistical analyses, the size data correlation coefficient rofthese distributions was were log-transformed. Mean hatching time, calculated in a regression analysis. The null hatchability and size at T0, T1 and T2 were com- hypothesis "r= 0"was tested as a t-test. Analysespared among and within mating system in a nested of covariance (ANCOVA) were performed to com- analysis of variance (ANOVA) with unequal pare the regressions between the two mating sys- sample size. To analyse survival data, we perfor- tems and also among different tanks in cross- med a nested ANOVA on the survival logit using fertilization. a variance proportional to a binomial variance with In Experiment 2, hatching time, hatchability, a quasi-likelihood model (McCullagh and Nelder, size at birth, survival and size at 14 and 30 days 1983). Except for this last ANOVA, all the analysis were compared between selfed and outcrossed F2 in Experiments 1 and 2 were performed following offspring. The partition of snails is the same as in Sokal and Rohlf (1981). Experiment 1, except that 10 snails only were used for self-fertilization. The capsules were collected Inbreedingdepression estimates every 2 days for 16 days. Only a quarter of the In outcrossed capsules, randomly chosen among Experiments 1 and 2, several fitness components those laid, were studied, because of the high pro- were estimated at different stages of the life cycle ductivity of the outcrossing groups. The number of snails (egg-laying rate of the parents, survival of eggs per capsule Nec was counted and each and size of the offspring). When the parameters capsule was then checked every 2 days during 2 measured were significantly different between self- weeks after hatching of the first snail (time ti).We fertilization and cross-fertilization, they were used noted: to estimate the inbreeding depression (see Lande (1) the hatching time tforall individuals and and Schemske, 1985; Charlesworth and Charles- the hatchability S0. for all capsules. worth, 1987):
Soc °(Noc/Nec) * 100 1— Wi(j)/wo(j) where N0 is the number of hatchlings per withw1(j)= meanperformance in self-fertilization capsule. for parameter jandw0(j)= meanperformance in The mean hatching time Tm for each cross-fertilization for the same parameter. capsule was: The overall difference between the two mating systems was calculated as: k /k ii k i1 j=I d1fl Wi(j)/wo(j) j=1 with n3snailshatching at time i, and k = numberof hatching observations. with k =numberof parameters considered. MATING SYSTEMS IN LYMNAEA PEREGRA 171
RESULTS performed (table 2) which showed that the hypothesis of single regression could not be accep- Experiment 1 ted. The regressions for cross-fertilization and self- Themean number of eggs per capsule (table 1) is fertilization are presented in figs. 1 and 2 respec- greater in cross-fertilization than in self-fertiliz- tively. ation (t315= 16.9, P<0.01). The number of eggs and the number of egg capsules per snail and per 2 day are given in table 1. The linear correlation Experiment coefficients between the number of eggs and the Numerousselfed capsules did not hatch and sur- dry weight of capsules are given in table 2. As 90 vival at T1 and T2 was very low. Thus, for the size per cent of the selfed capsules have less than 15 analysis, all the data from self-fertilization were eggs (60 per cent in cross-fertilization), we also taken into account, whereas only one randomly computed the correlation coefficient for these cap- chosen individual per outcrossed capsule among sules separately. All the correlation coefficients are the three measured was considered. Hatching time significantly different from zero (table 2). and size are given in table 3. The results of the The ANCOVA shows a significant difference nested ANOVA comparing these parameters when regressions for the two mating systems are between the two mating systems showed that there compared (table 2). However, the difference is not are no significant differences at any stage (table significant when capsules with less than 15 eggs 4). F-values for the subdivisions within systems are considered. In this case, as for data from cross- are significant at T1 and T2 showing that there is fertilization, the second step of ANCOVA was some variance between the cross-fertilization groups. Survival data are given in table 3. Out- crossed snails survived significantly better than Table 1 Egg capsule characteristics and oviposition rates. n selfed ones (ANOVA, table 5). There are no isthe mean number of eggs per capsule and u(n)its standard deviation differences between the outcrossed groups.
Cross-F Self-F Mating system Inbreedingdepression estimates Numberof eggs n 1354 759 871 793 Wecalculated the results of self-fertilization versus /capsule o-(n) cross-fertilization snails. Data for hatching time Numberof eggs 364 185 and size were not included, as there was no sig- snail/day nificant difference between mating systems. 1 —d3 Number of egg capsules 027 025 and d values for each stage are given in table 3. /snail/day The overall coefficient estimating the inbreeding
Table 2 Correlation coefficients between the number of eggs per capsule and the capsule weight and analysis of covariance F-values. The number of degrees of freedom is given in italics below each value ANCOVA F-values
Correlation Homogeneity Single coefficient of slopes regression
All capsules 0.891*** 294*** 314 1,312 Capsules<15 eggs 0.789*** 14 ns 17.6*** 222 1,220 1,221 Cross-fertilization capsules 0901*** 24 ns 352*** 197 3,191 3,194 Self-fertilization capsules 0879*** 115
* = P<0.05. **=P<0.01. ***=P<0.001. ns =non-significant. 172 P.JARNE AND B. DELAY
Log w Log w 2.00 2.00
1.00 1.00
0.00 0.00
—1.00 —1.00
—2.00 —2.00
—3.00 —3,00 0. 1.00 2.00 3.00 4.00 Log n Log n Figure 1Variation of the logarithm of the dry weight according Figure 2 Variation of the logarithm of the dry weight according to the logarithm of the number of eggs in cross-fertilization. to the logarithm of the number of eggs in self-fertilization. n is the number of eggs per capsule and w is the dry weight The equation of the regression line (dashed line) is: y = (in mg) of a capsule. The equation of the regression line 0695x—1580.The regression line for cross-fertilization is: y=0888x—2118. data is superimposed as a continuous line.
Table 3 Results of cross-fertilization and self-fertilization for the two experiments and their standard deviation in italics. 1 —dis the relative performance of selfed versus outcrossed hatchtings. d and 1 —dare calculated over all stages showing a significant difference between the mating systems. T0 is the date corresponding to the mean hatching time. T1 and T2 are respectively 14 and 30 days after T0
Cross-F Self-F 1 — , d1 Number of eggs/snail/day 364 185 0508 0492 T0 Hatching time T (days) 1456 F37 1455 233 — — Hatchability S0 0793 0198 0307 0266 0387 0•633 Size (mm) 1723 0117 1671 0130 — —
T1 Survival S, 0770 0195 0409 0413 0531 0469 Size (mm) 2981 0772 2405 0648 — —
T2 Survival S2,, 0837 0226 0478 0467 0571 0429 Size (mm) 4315 0976 3634 1212 — — Overall 0060 0940 depression is 0940 one month after hatching (table and self-fertilization to find out whether there was 3). a different weight investment in capsules in L. peregra. In all cases, the linear correlation coefficient DISCUSSION between the number of eggs and the dry weight of a capsule was high and significantly different from Insome plant species, seed weight is a goodzero. This indicates that the correlations are indicator of seed viability and seedling growth rate effectively linear. The ANCOVA showed a (Stanton, 1984), although Stevens and Bougourd difference between outcrossed and selfed capsules. (1988) did not find such a relation in Allium However, when the egg capsules with more than schoenoprasum. We compared cross-fertilization 15 eggs are discarded, the hypothesis of MATING SYSTEMS IN LYMNAEA PEREGRA 173
Table 4 Results of the nested ANOVA on hatching time and size between and within the two mating systems. The number of degrees of freedom is given in italics below each value
Source of Mating Subdivisions variation system within systems Residuals
Hatching time 0272 ns 0105 ns 1 4 94
T0 Size 3961 ns 2119 ns 1 4 101
T1 Size 1837 ns 8.078*** 1 4 99
T2 Size 1354 ns 3.522* 1 4 85 = P<0•05. ***=P<0.00l. ns =non-significant.
Table S F-values obtained in the nested ANOVA comparing in the case of the smallest capsules (e.g., below survival between and within the two mating systems. The five eggs). For more than 15 eggs, selfed capsules number of degrees of freedom is given in italics below each may be lighter than outcrossed. Even if there is an value actual slight difference in capsule weight, the eggs Subdivisions of one mating system are not systematically Time Mating system within systems heavier. It seems that the differential survival is not due to a differential weight investment in eggs T0 79.677*** 0680 ns and that egg weight is a poor indicator of viability. 1,119 1,115 In the second experiment, there was no sig- T1 32.774*** 2110 ns nificant difference in hatching time. The higher 1,108 1,104 mortality does not seem to be due to a too short T2 79.057*** 3880 ns development time within the selfed capsules. There 1,920 1,880 was also no apparent difference in size at birth, T1 ***= P
ROLLINSON, D., KANE, R. A. AND LINES, J. R. L. 1989. An STANTON, VI. L. 1984. Seed variation in wild radish: effects of analysis of fertilization in Bu!inuscernicus(Gastropoda: seed size on components of seedling and adult fitness. Planorbidae). J. Zoo!., London, 217,295—310. Ecology, 65, 1105-1112. SCHEMSKE, D. W. AND LANDE, R. 1985. The evolution of STEVENS, J. P. AND BOUGOURD, S. M. 1988. Inbreeding self-fertilization and inbreeding depression in plants. II. depression and the outcrossing rate in natural populations Empirical observations. Evolution, 39, 41—52. of Allium schoenoprasum L. (Wild chives). Heredity, 60, SELANDER, R. K. AND KAUFMAN, D. w. 1973. Self-fertilization 257—26 1. and genetic population structure in a colonizing land snail. VIANEY-LIAUD, M. 1989. Growth and fecundity in a black- Proc.Nat!Acad. Sci. USA, 70, 1186-1190. pigmented and an albino strain of Biornphalaria g!abrata SELANDER, R. K., KAUFMAN, D. W. AND RALIN, R. S. 1975. (Gastropoda: Pulmonata). Malacological Review, 22, 25- Self-fertilization in the terrestrial snail Rumina decollata. 32. The Veliger, 16, 265-270. SOKAL, R. R. AND ROHLF, F. .i. 1981. Biometry, 2nd edn. Freeman.