Luc Madec

Multiple paternity and postcopulatory sexual selection in a hermaphrodite: what influences sperm precedence in the garden snail Helix aspersa?

Sperm competition has been studied in many gonochoric animals but little is known about its occurrence in simultaneous hermaphrodites, especially in land snails. The reproductive behaviour of the land snail Helix aspersa involves several features, like multiple mating, long‐term sperm storage and dart‐shooting behaviour, which may promote sperm competition. Cryptic female choice may also occur through a spermatheca subdivided into tubules, which potentially allows compartmentalized sperm storage of successive mates. In order to determine the outcome of postcopulatory sexual selection in this species, we designed a cross‐breeding experiment where a recipient (‘female’) mated with two sperm donors (‘males’). Mates came from either the same population as the recipient or from a distinct one. To test for the influence a recipient can have on the paternity of its offspring, we excluded the effects of dart shooting by using only virgin snails as sperm donors because they do not shoot any dart before their first copulation. We measured the effects of size of mates as well as time to first and second mating on second mate sperm precedence (P2; established using microsatellite markers). Multiple paternity was detected in 62.5% of clutches and overall there was first‐mate sperm precedence with a mean P2 of 0.24. Generalized linear modelling revealed that the best predictors of paternity were the time between matings and the time before first mating. Overall, both first and second mates that copulated quickly got greater parentage, which may suggest that postcopulatory events influence patterns of sperm precedence in the garden snail.

Spatial analysis of allozyme and microsatellite DNA polymorphisms in the land snail Helix aspersa (Gastropoda: Helicidae)

The genetic structure of the land snail Helix aspersa was investigated for 21 populations collected along a road located in the polders of the Bay of Mont‐Saint‐Michel (Brittany, France), following a sampling scheme the area of which did not exceed 900 m in length. A total of 369 individuals were genotyped for five enzymatic markers and seven microsatellite loci. We used sequential hierarchical F‐statistics at different spatial scales and spatial autocorrelation statistics to explore recent historical patterns involved in the observed genetic distribution. Whatever the statistics used, congruent levels of spatial genetic substructuring across loci were demonstrated, excepted for one allozyme locus. Overall spatial genetic arrangement matched in a substantial fashion theoretical predictions based on the limited dispersal power of land snails. Positive autocorrelation over short‐distance classes may result from the development of genetically distinct patches of individuals organized in family‐structured colonies. Therefore, spatial signatures of average I correlograms can be viewed as the expression of a stepping‐stone model of population structure, sometimes involving external migrational events. Overall, the revealed pattern of population subdivision on a microgeographical scale was suggestive of a neighbourhood structure. Finally, microsatellite loci are especially suitable for the detection of small genetic clustering, and combining different classes of markers offers the potential to gain further insight into the description of spatial genetic variability over short temporal and geographical scales.

Microspatial genetic structure in the land snail Helix aspersa (Gastropoda: Helicidae)

The microspatial genetic structure of allele frequencies at seven isozyme loci was examined for 15 populations of the land snail Helix aspersa sampled in a village from Brittany (north-western France). Spatial heterogeneity of allele frequencies was highly significant (P < 0.001). Fixation indices reflected nonrandom mating within neighbourhoods and a slight but consistent differentiation between colonies (FST=0.044; P < 0.01). Analyses of gene flow or genetic distances failed to reveal a significant relationship with geographical distance, probably because of the complexity of environmental heterogeneity. However, matrix comparisons between genetic distances and connectivity networks among adjacent colonies (Gabriel-connected graph) yielded a significant correlation in every case, indicating a ‘step-by-step’ relationship between neighbouring localities. Moreover, most of the allozymes were spatially structured and showed (i) a gradual isolation of colonies with increasing geographical distances, and (ii), for some correlogram profiles, a circular gradient illustrating a multidirectional colonization of the village. The probable existence of disperser individuals allowed us to suggest a metapopulation model which would explain the maintenance of such animals in fragmented habitats where anthropogenic disturbances and extinction/recolonization events are commonly observed.

Shell shape of the land snail Cornu aspersum in North Africa: unexpected evidence of a phylogeographical splitting

Anatomical and molecular characters used to differentiate populations of the land snail Cornu aspersum (Helix aspersa) exhibit, in the western Mediterranean, definite and concordant patterns of correlation with geography. Scenarios involving Pliocene geological changes and postglacial expansion during the Pleistocene were proposed in previous studies to account for the establishment of this geographical structure. In the present work, we have performed a spatial analysis of variation in shell morphometrics, after the partitioning of the overall variation into size and shape components by means of a principal component-based approach (Cadima and Jolliffe, 1996). In order to know if the same historical events have also structured shell variation, the analysis includes all the populations from North Africa which were investigated for anatomical and molecular surveys. Contrary to shell size, which shows a significant spatial heterogeneity essentially related to environmental pressures, variation in shell shape components splits the populations according to a geographical pattern reflective of hypotheses suggested for molecular markers and genital anatomy. This implies that the selective forces often invoked to explain spatial changes in shell shape are not the deciding factors in the present case. Moreover, within each of the two geographical clusters defined, Mantel correlograms show that the similarity between populations declines according to an isolation by distance model. Because of the different allometric relationships between shell size and genitalia measurements in Western and Eastern entities of North Africa, mechanical constraints, possibly leading to a precopulatory isolation in the contact zone, are involved.

INBREEDING AND FITNESS IN THE FRESHWATER SNAIL LYMNAEA PEREGRA: AN EVALUATION OVER TWO GENERATIONS OF SELF-FERTILIZATION

Inbreeding depression was estimated from an outbreeding population of the freshwater snail Lymnaea peregra, on the basis of two successive generations of enforced selling and outcrossing, and 70 maternal lineages. Outcrossing was analyzed under two treatments, groups of two and five individuals. The fitness parameters measured included fecundity, growth, and survival. In the first generation, we contrasted three treatments (selfers vs. paired outcrossers and group outcrossers). Very similar results were obtained between the two outcrossing treatments. A strong self‐fertilization depression (which includes parental fecundity and progeny fitness) was detected in the selling treatment (about 90%). In the second generation, there was again marked evidence for self‐fertilization depression, with the highest contributions coming from parental fecundity and progeny hatching rate. Our results suggest that the decreased parental fecundity is a consequence of the mating system in the previous generation, although the role of partial self‐incompatibility and the copulation behavior could not be ruled out. Hatching rate and early survival data are suggestive of purging of lethal mutations. Significant variation in fitness among selfing lineages was found for most fitness traits. Our experimental design also allowed to test for interactions among fitness loci. Only one trait of the nine studied behaved as expected under synergistic interactions. However we cannot rule out some purging during the experiment, which could have biased results towards linearity. Inbreeding depression was also inferred from the change of inbreeding level across generations in the same population. We obtained a value similar to the experimental estimate.

Tracing the invasion of the Mediterranean land snail Cornu aspersum aspersum becoming an agricultural and garden pest in areas recently introduced.

This study is the first on the genetics of invasive populations of one of the most widely spread land mollusc species known in the world, the “Brown Snail” Cornu aspersum aspersum. Deliberately or accidentally imported, the species has become recently a notorious pest outside its native Mediterranean range. We compared the spatial structure and genetic variability of invasive (America, Oceania, South Africa) versus native populations using five microsatellite loci and mitochondrial (Cyt b and 16S rRNA) genes as a first step towards (i) the detection of potential source populations, and (ii) a better understanding of mechanisms governing evolutionary changes involved in the invasion process. Results based on multivariate analysis (Discriminant Analysis of Principal Components), Bayesian statistical inference (Clustering, Approximate Bayesian Computation) and demographic tests allowed a construction of the introduction pathways of the species over recent centuries. While emigrants originated from only one of the two native lineages, the West one, the most likely scenario involved several introduction events and “source switching” comprising (i) an early stage (around 1660) of simultaneous introductions from Europe (France, Spain) towards Oceania (New Zealand) and California, (ii) from the early 18th century, a second colonization wave from bridgehead populations successfully established in California, (iii) genetic admixture in invasive areas where highly divergent populations came into contact as in New Zealand. Although these man-made pathways are consistent with historical data, introduction time estimates suggest that the two putative waves of invasion would have occurred long before the first field observations recorded, both in America and in Oceania. A prolonged lag period as the use of an incorrect generation time could explain such 100–150 years discrepancy. Lastly, the contrasting patterns of neutral genetic signal left in invasive populations are discussed in light of possible ways of facing novel environments (standing genetic variation versus new mutation).

Spatial structure of shell polychromatism in populations of Cepaea nemoralis: new techniques for an old debate

A conspicuous shell polychromatism is observed in colonies of Cepaea nemoralis from western France (Brittany). The present study is intended to search for a spatial structure of shell features at this scale and to infer evolutionary processes from the observed patterns. We used a database of morph frequencies (six composite phenotypes were retained) measured on 213 samples regularly distributed on the whole studied area. Data analysis was based on two distinct multivariate methods leading to the following steps: (i) to search for a structure without reference to environmental conditions with a method (global principal components analysis: GPCA) which takes into account the spatial information by means of a neighbouring relationship between sampling points (Delaunay triangulation); (ii) to test the structuring power of environmental conditions by means of two explanatory factors (distance from the sea, altitude) involved in a redundancy analysis (RDA); (iii) to search for a spatial structure using residuals of the previous analysis, ie, after removing effects of environmental conditions. Global covariance accounted for 26.4% of the total variance, leading to a highly significant autocorrelation for each phenotype (step 1). Geographical mapping of factorial scores resulting from global analysis showed a well structured littoral zone and a strong southern-northern inland differentiation. Sixteen percent of the total variance was expressed in RDA but all morphs were not equally concerned. After removing environmental effects, a significant spatial structure still remains but was essentially caused by random processes. We argue for the importance of these last phenomena.

Population genetic structure in a human-disturbed environment: a case study in the land snail Helix aspersa (Gastropoda: Pulmonata).

Local patterns of genetic variation were analysed in the land snail Helix aspersa for 32 populations sampled within a patchy agricultural landscape: the polders of the Bay of Mont-Saint-Michel (France). This investigation examined the allele frequencies at four enzymatic markers and five microsatellite loci through the genotyping of 580 individuals. A strongly significant population genetic substructuring (mean FST=0.088, P<0.001) was found at the scale of the whole polders area (3050 ha) and both categories of markers displayed a similar magnitude of spatial genetic differentiation. We did not find any obvious effects of habitat fragmentation on the distribution of genetic variability. Despite the reality of habitat patchiness and environmental instability (related to farming practices), an isolation by distance process was clearly depicted, although selective pressures cannot be ruled out for one enzymatic locus. Overall, genetic drift, along with occasional long-distance episodes of gene flow, was presumably the most likely evolutionary force that shaped the observed pattern of genetic variation.

Selfing and biparental inbreeding: a mating system analysis in Lymnaea peregra (Gastropoda: Lymnaeidae)

The mating system was analysed in three populations of Lymnaea peregra, under the mixed-mating model (MMM) and the effective selfing model (ESM), using progeny array data and allozyme markers. The results revealed variation among populations for the outcrossing rate (tm ranged from 0.646 to 0.983). This finding modifies the earlier classification of L. peregra as a predominantly outcrossing species. The occurrence of biparental inbreeding and population substructure in one of three populations was suggested from positive (tm—ts) under the MMM, covariation of the effective selfing rate with parental fixation index, as well as the great difference between the effective selfing rate of inbred and outbred parents under the ESM. The within-population distribution of the outcrossing rates varied extensively and may involve different patterns of selection and levels of inbreeding depression among the populations studied. Consistently, the estimation of correlated matings showed different levels of the correlation of selfing within progenies in the three populations. Levels of multiple paternity for the outcrossed progeny were positively associated with higher population outcrossing rates, which may reflect the propensity of preferential outcrossers to copulate actively.

Movement propensity and ability correlate with ecological specialization in European land snails: comparative analysis of a dispersal syndrome

Intra- and interspecific differences in movement behaviour play an important role in the ecology and evolution of animals, particularly in fragmented landscapes. As a consequence of rarer and generally more fragmented habitat, and because dispersal tends to disrupt benefits brought by local adaptation, theory predicts that mobility and dispersal should be counter-selected in specialists. Using experimental data and phylogenetic comparative tools, we analysed movement propensity and capacity, as well as dispersal-related phenotypic traits, in controlled conditions in 20 species of European land snails from the Helicoidea superfamily. Costs of movement in terrestrial gastropods are among the highest in animals, which make them a potentially powerful model to test these predictions. Habitat specialists were indeed less likely to cross a boundary between a familiar and an unfamiliar substrate than generalists. They also had smaller feet, after accounting for size. Furthermore, exploring specialists were slower than generalists and had more tortuous trajectories, leading them to stay closer to the familiar patch. Movement traits were generally evolutionary labile, but some were constrained by body size, a phylogenetically conserved trait. High specialization and low-dispersal ability are two traits often considered to increase species vulnerability to fragmentation, climate changes and extinction. This study confirms they should not be considered separately, due to their integration in a dispersal syndrome. Therefore, specialist species face double penalty under habitat loss and other environmental changes, making them more vulnerable to extinction and contributing to the biotic homogenization of communities.