RESEARCH NOTE Life-history variation and breeding system in the hermaphroditic land snail Succinea putris (Pulmonata: Succineidae) Lobke Dillen1, Kurt Jordaens1 and Thierry Backeljau1,2 1
Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium; and 2 Royal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium
Pulmonate snails and slugs are simultaneous hermaphrodites, although some species have a short ‘male’ phase during which only sperm are produced (i.e. they are ‘slightly protandric’, see Jordaens, Dillen & Backeljau, 2007). Pulmonates are well known for their wide variation in life-history characters, in traits such as fertility and fecundity, which are influenced by a number of genetic and environmental factors (reviewed in Jordaens et al., 2007). Among the factors affecting life history in hermaphroditic organisms, the breeding system (i.e. selffertilization, outcrossing or a combination of both) is one of the most important, since it is intimately related to in- and outbreeding depression and sex allocation (Jordaens et al., 2007). Therefore, in order to study the evolution of life-history characters in hermaphrodites, a sound knowledge of the breeding system is indispensable. In theory, simultaneously hermaphroditic snails are capable to reproduce both by outcrossing and self-fertilization (e.g. Heller, 2001). However, the breeding system varies greatly within and among basommatophoran (reviewed by Duncan, 1975; Brown & Richardson, 1988; Vianey-Liaud, 1990; Jarne, Vianey-Liaud & Delay, 1993; Jarne & Sta¨dler, 1995; Dillon, 2000) and stylommatophoran (reviewed by Duncan, 1975; Selander & Ochman, 1983; Brown & Richardson, 1988; Heller, 1993) species. Heller (1993) lists 18 pulmonate genera (of 12 families) that are capable of self-fertilization. Basommatophoran species in particular seem to have a mixed breeding system (i.e. they combine outcrossing and selffertilization) and several of these species show significant geographical variation in their breeding system (Jordaens et al., 2007). Nevertheless, several stylommatophoran genera (e.g. Arion, Cochlicopa; see Jordaens et al., 2007 and references therein) contain, besides species that appear to be strict outcrossers or strict self-fertilizers, additional species that have a mixed breeding system (reviewed in Jarne & Auld, 2006). A case in point is the stylommatophoran family Succineidae in which self-fertilization has been demonstrated in eight species of five genera, viz. Succinea grosvenori, Succinea unicolor, Succinea campestris, Omalonyx feline, Oxyloma retusa, Oxyloma salleana, Catinella sp. and Quickia spurca (Patterson, 1970). Nine other species (not listed) did not reproduce by self-fertilization (Patterson, 1970). So far, the breeding system of Succinea putris (Linnaeus, 1758) is not known although it is the most common and widely distributed European succineid species. The mating behaviour of the species is relatively well known (Rieper, 1912; Hecker, 1965; Jordaens, Pinceel & Backeljau, 2005; Dillen et al., 2008; Dillen, Jordaens & Backeljau, in press). Mate choice is random with respect to shell size, yet, in pairs where the partners are of unequal shell size, most commonly the smaller individual mounts the shell of the lower, larger individual during courtship. Penes are intromitted reciprocally and, in general, sperm are also transferred reciprocally (Jordaens et al., 2005). Eggs are laid in gelatinous translucent Correspondence: L. Dillen; email:
[email protected]
masses from April until the end of September (Rieper, 1912; Hecker, 1965). Although copulations are easily observed in natural populations during the breeding season, or under laboratory conditions, the exchange of sperm is no proof of outcrossing. Thus, it is unknown whether the species is capable of self-fertilization. Here, we studied the variation in life-history characters in S. putris under controlled laboratory conditions, with special emphasis on the effects of the breeding system on life-history variation. Approximately 100 recently hatched snails (shell height ,3 mm) were collected from a population in Wilrijk (Belgium) in November 2005. The snails were kept isolated in transparent plastic containers (7.5 cm diameter 5.5 cm height) of which the bottom was covered with moist paper during the first 2 months. Afterwards, the bottom was covered with a layer of moist compost to which calcium carbonate powder was added. The containers were placed in a climatecontrolled room (208C) under a light:dark cycle of 18 h light:6 h dark. Lettuce, carrots and leaves of Populus x canadensis and Alnus glutinosa were given as food ad libitum. Every second day, containers were cleaned and fresh food and leaves were provided. The compost was changed every 2 months. Individuals of S. putris with a shell height above 9 mm are able to copulate ( personal observations). Most individuals were large enough for use in a mating trial after 4 months. After 5 months the remaining individuals had also reached the required body size and these were used for another mating trial. During a trial, the sexually mature individuals were put together in a large container. Then, approximately one-third of the individuals were drawn randomly from the pool, while the other individuals were allowed to copulate. Copulating pairs were gently removed from the container to prevent disturbance by other snails. After copulation, both partners were kept isolated as described above. Thus, there were a total of 46 single-mated individuals (i.e. 23 pairs) and 43 unmated individuals of which the life history was followed. Every day or every second day the containers were checked for the presence of egg clutches. Egg clutches were weighed to the nearest 0.0001 g using an electronic Sartorius balance. The eggs of each clutch were counted (¼ clutch size) and checked for the presence of multiple embryos under a binocular microscope. Subsequently, the egg clutches were transferred to containers containing wet paper and kept in the same climate-controlled room. Every day or every second day the clutches were checked for hatched juveniles, which were counted and transferred to other containers. If eggs failed to develop within 45 days they were considered to be unfertilized. The hatching period was defined as the number of days between the clutch being laid and the day when 50% of the eggs hatched, and hatching success as the proportion of successfully hatched eggs. All snails were followed until they died, after which their shell height and shell width were measured to the nearest 0.01 mm with calipers (except for a few shells that were broken during dissection), and shell volume was calculated as V ¼ (4/3)
Journal of Molluscan Studies (2009) 75: 311–313 # The Author 2009. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved
RESEARCH NOTE ( pa 2b) (where a is 0.5 shell breath, and b is 0.5 shell height). The two groups did not differ in their mean shell volume (t-test: 21.43; df ¼ 79; P ¼ 0.16). The reproductive period was defined as the period starting from the day a first clutch was produced until the last egg clutch was laid. The period between the day when the last egg clutch was laid and the day when the individual died was defined as the senescence period. Our study showed that most (42/46) of the single-mated individuals produced viable eggs with a mean number of eggs of 282 (Table 1). Four individuals (9%) did not produce eggs, although they survived for several months after copulation. In two previous studies, respectively, 14% (Jordaens et al., 2005) and 9% (Dillen et al., 2008) of the matings involved unilateral sperm transfer in S. putris, so the four individuals of this study that did not produce eggs probably did not receive sperm during copulation. Succinea putris shows a large variation in several life-history characters, such as fecundity, fertility and hatching time (Table 1). Clutch size in our study varied between 1 and 176 eggs and the mean hatching time was c. 16 days. Hecker (1965) reported variable clutch sizes of 40– 50 eggs (maximum 95 eggs) and a hatching time of c. 18 days.
Fro¨mming (1954) observed clutch sizes of 50 –100 eggs with a maximum of 136 eggs. Rieper (1912) reported clutch sizes up to 95 eggs and a hatching time of c. 18 days. Hatching time strongly depended on the temperature and varied between 10 days (high temperatures) and 3 weeks (low temperatures) (Rieper, 1912; Fro¨mming, 1954). Because the outcrossing individuals were only allowed to copulate once, we do not know whether one copulation is enough to fertilize all the eggs. The mean hatching success of consecutive clutches was very similar (e.g. first clutch: 70%, second: 85%, third: 80%, fourth: 78%, fifth: 75%, sixth: 53%, seventh: 93% and eighth: 76%) and the mean hatching success of the final clutch was 68%. This may suggest that individuals were not sperm-depleted when they laid their last clutch. Only 14 out of 43 unmated individuals produced eggs (with mean number of eggs of 2.9). The mean hatching success of single-mated individuals was 79%, whereas only 1 egg out of the 41 eggs (2.4%) of unmated individuals hatched (Table 1). We also observed that the mean egg-laying period and mean clutch size were much lower, and the mean number of days between two successive clutches was higher, in unmated individuals compared to single-mated individuals (Table 1). Thus, our results suggest that S. putris is a predominant outcrosser. Our results are in agreement with a previous allozyme study in four Belgian populations that showed significant heterozygote deficiencies in some populations, but since deficiencies were not consistent over all loci they were attributed to genetic drift and not to occasional self-fertilization (Jordaens et al., 2006). Our study also shows that (occasional) self-fertilization does not account for the high variation observed in many life-history parameters in S. putris. In theory, the major disadvantage of self-fertilization is the occurrence of inbreeding depression, i.e. the depressed fitness of offspring from self-fertilizing individuals compared to that of outcrossing individuals. In addition, self-fertilizers often show a reduced fecundity. The combined effects of inbreeding depression and a reduced fecundity in self-fertilizers compared to outcrossers are referred to as self-fertilization depression (Jarne et al., 1991). Our results suggest strong self-fertilization depression in S. putris, since unmated individuals showed a reduced fecundity (i.e. 1% of that of outcrossers) and a reduced fertility (i.e. 2.4% vs 79% in outcrossers). Similar results were obtained for the predominant outcrossing land snails Arianta arbustorum (Chen, 1993, 1994; Chen & Baur, 1993) and Triodopsis albolabris (McCracken & Brussard, 1980). Also, the shorter egg-laying period, the larger interval between two successive clutches and the lower egg weight in unmated individuals compared to single-mated conspecifics may suggest self-fertilization depression in unmated individuals. An hermaphroditic individual that prefers to outcross, but that has the potential of self-fertilization, faces a dilemma in the absence of a partner: should it wait for a partner for outcrossing or should it self-fertilize? Theory predicts an optimal delay of reproduction that balances the potential benefit of outcrossing and the cost of delaying the onset of reproduction and which has evolved as a function of inbreeding depression, resource reallocation and survival (Tsitrone, Duperron & David, 2003). The observation that hermaphroditic individuals start to reproduce by self-fertilization later than paired or grouped individuals start to reproduce by outcrossing, and which is not an inbreeding effect, is called ‘delayed selfing’. Many unmated individuals of S. putris did not reproduce in our experiment, despite the fact that they were followed during their whole life span, and those that laid eggs showed strong self-fertilization depression, suggesting that delayed selfing is not a likely strategy in S. putris. Finally, in single-mated individuals we observed several clutches (i.e. in 60 out of 262 clutches or 23%) that contained
Table 1. Descriptive reproductive statistics of single-mated and unmated individuals of Succinea putris. Once-mated
Unmated
No. of individuals
46
Mean shell volume (mm3)
616.6 + 32.7
43 707.9 + 58.9
No. of individuals producing eggs
42
14
5.4 + 0.6
–
1
–
17
–
Mean length of egg laying period (days)
29.3 + 2.7
8.5 + 3.4
Maximum length of egg laying period
82
39
Length of senescence period (days)
0– 32
2– 73
Mean no. of days between two clutches
5.3 + 0.3
12.4 + 4.1
Minimum no. of days between two
0
0
38
37
Total no. of eggs (fecundity)
11,861
41
Total no. of clutches
262
22
Mean no. of clutches/individual
6.24 + 0.6
1.6 + 0.3
Range of no. clutch/individual
1– 23
1– 5
Mean no. eggs/individual
282 + 32
2.9 + 0.71
Range of no. eggs/individual
4– 821
1– 10
Mean clutch size
45.3 + 2.1
1.9 + 0.4
Range of clutch size
1– 176
1– 10
Mean clutch weight (g)
0.15 + 0.11
0.014 + 0.003
Mean egg weight (g)
0.004 + 0.0001
0.009 + 0.001
Total hatching success (fertility)
0.79
0.02
Mean hatching success per individual
0.78 + 0.03
0.04 + 0.04
Mean hatching success per egg clutch
0.79 + 0.02
0.02 + 0.02
Mean hatching time (days)
15.6 + 0.15
16
Reproductive period Mean no. of days between copulation and egg laying Minimum no. of days between copulation and egg laying Maximum no. of days between copulation and egg laying
(days)
clutches Maximum no. of days between two clutches Clutch characteristics
Hatching success and hatching time
Means are given +standard error.
312
RESEARCH NOTE HECKER, U. 1965. Zur Kenntnis der mitteleuropa¨ischen Bernsteinschnecken (Succineidae). Archiv fu¨r Molluskenkunde, 94: 1– 45. HELLER, J. 1993. Hermaphroditism in molluscs. Biological Journal of the Linnean Society, 48: 19– 42. HELLER, J. 2001. Life history strategies. In: The biology of terrestrial molluscs (G.M. Barker ed.), pp. 413–445. CABI Publishing, New York. JARNE, P. & AULD, J.R. 2006. Animals mix it up too: the distribution of self-fertilization among hermaphroditic animals. Evolution, 60: 1816– 1824. JARNE, P. & STA¨DLER, T. 1995. Population genetic structure and mating system evolution in freshwater pulmonates. Experientia, 51: 482–497. JARNE, P., FINOT, L., DELAY, B. & THALER, L. 1991. Self-fertilization versus cross-fertilization in the hermaphroditic freshwater snail Bulinus globosus. Evolution, 45: 1136– 1146. JARNE, P., VIANEY-LIAUD, M. & DELAY, B. 1993. Selfing and outcrossing in hermaphrodite freshwater gastropods (Basommatophora): where, when and why. Biological Journal of the Linnean Society, 49: 99– 125. JORDAENS, K., PINCEEL, J. & BACKELJAU, T. 2005. Mate choice in the hermaphroditic land snail Succinea putris (Stylommatophora: Succineidae). Animal Behaviour, 70: 329– 337. JORDAENS, K., DE WOLF, H., VAN HOUTTE, N., VANDECASTEELE, B. & BACKELJAU, T. 2006. Genetic variation in two land snails, Cepaea nemoralis and Succinea putris (Gastropoda, Pulmonata), from sites differing in heavy metal content. Genetica, 128: 227–239. JORDAENS, K., DILLEN, L. & BACKELJAU, T. 2007. Effects of mating, breeding system and parasites on life history variation in hermaphrodites: pulmonate gastropods (Mollusca). Animal Biology, 57: 137–195. McCRACKEN, G.F. & BRUSSARD, P.F. 1980. Self-fertilization in the white-lipped snail Triodopsis albolabris. Biological Journal of the Linnean Society, 14: 429 –434. PATTERSON, C.M. 1970. Self-fertilization in the land snail family Succineidae. Journal de Conchyliologie, 108: 61–62. RIEPER, H. 1912. Studien an Succinea. Annales de la Socie´te´ Royale Zoologique et Malacologique de Belgique, 47: 125– 191. SELANDER, R.K. & OCHMAN, H. 1983. The genetic structure of populations as illustrated by molluscs. Isozymes: Current Topics in Biological and Medical Research, 10: 93–123. TSITRONE, A., DUPERRON, S. & DAVID, P. 2003. Delayed selfing as an optimal mating strategy in preferentially outcrossing species: theoretical analysis of the optimal age at first reproduction in relation to mate availability. American Naturalist, 162: 318– 331. VIANEY-LIAUD, M. 1990. Biologie de la reproduction de Biomphalaria glabrata (Gaste´ropode: Pulmone´). The`se de Doctorat d’Etat, Universite´ Montpellier II, France.
eggs with multiple embryos (up to 15 embryos per egg). Although we cannot exclude that such eggs represent developmental abnormalities, we found in six out of these clutches a hatching success .1 (i.e. more hatchlings than eggs were counted). This shows that there were at least several eggs with multiple embryos where more than one of the embryos hatched. We also observed eggs with presumed multiple embryos in unmated individuals (i.e. in 8 out of 22 clutches, or 36%; these appeared similar to eggs with multiple embryos of mated individuals), but none of these eggs hatched. In summary, our study shows that the hermaphroditic land snail S. putris is a predominant outcrosser. The variation in lifehistory characters is very high and self-fertilization does not seem to account for part of this variation.
ACKNOWLEDGEMENTS This work was financed by a BOF NOI project (FA070400/3/ 1040) from the University of Antwerp and a ‘Krediet aan Navorsers’ (1.5.066.06) of the Fund for Scientific Research – Flanders (F.W.O.) to K.J. K.J. is a postdoctoral fellow of the F.W.O. We wish to thank Natalie Van Houtte for her help with the practical work and an anonymous referee for improving the manuscript.
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doi:10.1093/mollus/eyp031 Advance Access Publication: 6 June 2009
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