Isolation and characterization of highly polymorphic microsatellite markers from the marine invasive species Crepidula fornicata (Gastropoda: Calyptraeidae)

Many marine organisms have pelagic larvae and these are often important agents of dispersal and establishment of exotic species. The larval phase and the hermaphroditic reproductive system of the invasive marine gastropod Crepidula fornicata are crucial in determining the success of this species along the European coasts. To analyse these factors, we developed seven microsatellite loci specific to C. fornicata. On the same set of individuals, the microsatellite loci exhibited a greater level of polymorphism than previously studied allozyme markers and, thus, will permit fine‐scale analysis and larval pool studies.     

Infestation of the invasive mollusc <Emphasis Type="Italic">Crepidula fornicata</Emphasis> by the native shell borer <Emphasis Type="Italic">Cliona celata</Emphasis>: a case of high parasite load without detrimental effects

The temporal prevalence of the widespread boring sponge Cliona celata and its effects were analysed in a population of the invasive mollusc Crepidula fornicata. This mollusc produces extra shell material when infested with the endolithic sponge, suggesting that infestation may be detrimental for C. fornicata growth and/or reproduction. For 37 months, size, sex, female reproductive status and sponge-infestation stage were recorded for 300 individuals sampled every month in the Bay of Morlaix (France). In the 12,049 individuals examined, the prevalence of C. celata was high with a monthly average of 43.1% of the individuals hosting the sponge. The relative proportion of heavily infested individuals generally increased over time. Nevertheless, a cyclic decrease occurred every 10 months, suggesting putative episodes of mortality of heavily infested individuals. The gregarious behaviour of the mollusc seemed to promote the high prevalence of the sponge, which may propagate through contact between neighbouring C. fornicata individuals. Due to the sex-size relationship in protandrous C. fornicata, females were far more infested than males. We did not find evidence for a cost of producing extra shell material on somatic growth or on female fertility, and the boring sponge is unlikely to substantially affect the sex-change patterns in C. fornicata. The limited effects of the endolithic sponge on C. fornicata contrasts with the documented damage to some local species, including commercially exploited shellfish, suggesting that C. fornicata may alter the infestation dynamics in the surrounding native community. Dedicated studies are now needed to investigate the extent and mechanisms of these species interactions.     

Microsatellite evidence for sperm storage and multiple paternity in the marine gastropod Crepidula coquimbensis

In gregarious species with copulation and internal fertilization, male-male competition and female cryptic choice may affect reproductive success of both sexes. We carried out a molecular analysis to study paternity and sperm use by females in the protandrous marine brooding gastropod Crepidula coquimbensis. In the field, a single female inhabits an empty hosting shell with up to six males. This gregarious behavior may promote intra-brood multiple paternity if females can store sperm from several consecutive copulations with the surrounding males. To study female sperm usage, the males sharing shelters with five different adult females were collected and preserved for paternity analysis. Females were transported alive to the laboratory and isolated for six months. After that, an additional male was offered to each of the five study females. Once the females had laid capsules, a total of 528 embryos from the five females were assigned paternity based on five microsatellite loci. Paternity analysis showed that every male sharing the empty hosting shell of a female as well as the additional male were assigned fatherhood of embryos laid by this specific female. Females can thus use sperms from multiple males including sperms stored for at least six months. In addition, in four out of the five offspring arrays, a similar contribution of each male to the brood was observed, a pattern associated with the close relationship between the number of fathers observed and the effective paternity index calculated. These results contrast with those of paternity analyses carried out in another species of the same genus, C. fornicata which is characterized by a stacking behavior in which the closest male to the female achieves the highest reproductive success. Male reproductive success may be largely influenced by the aggregation pattern and male mating opportunities in the Crepidula complex, a hypothesis to be examined further by studying other species exhibiting different grouping behavior.     

Isolation and characterization of microsatellite loci in Acer opalus (Aceraceae), a sexually-polymorphic tree, through an enriched genomic library

An enrichment protocol was used to isolate and characterize microsatellite loci in Acer opalus, a Mediterranean tree species. Highly polymorphic microsatellite loci were required for paternity analyses in a population of this species. Eight microsatellite loci were amplified and a total of 87 alleles were detected in a sample of 142 individuals from one population, allowing the identification of each individual with a unique multilocus genotype. The paternity exclusion probabilities varied from 0.261 for locus Aop820 to 0.806 for locus Aop450, and the parent-pair exclusion probabilities varied from 0.433 for Aop820 to 0.940 for Aop450. The cumulative probabilities of exclusion for paternity and parentage of the eight loci were both higher than 0.999, supporting the usefulness of these microsatellite loci for future paternity and parentage analyses in A. opalus. Cross-species transferability was also assayed, supporting their potential use in other eight Acer species.     

Characterization of six microsatellite loci in Echium vulgare (Boraginaceae)

Echium vulgare is a tetraploid plant with a very low selfing rate in the field. We suspect that cryptic self incompatibility plays a role in this species. In order to show lower success of self pollen/selfed embryos, after pollination with a mixture of self and outcross pollen, a paternity analysis has to be done. For the purpose of such analysis we developed microsatellites in E. vulgare. In this study, we report on six microsatellite loci which are easy to score, polymorphic, with a number of alleles per locus ranging from two to eight and, therefore, suitable for paternity analysis.     

Nineteen new microsatellite DNA polymorphisms in pigtailed macaques (Macaca nemestrina)

Microsatellite loci known to be polymorphic in baboons (Papio hamadryas) and/or humans were tested in pigtailed macaques (Macaca nemestrina) from the Washington Regional Primate Research Center. Nineteen polymorphisms were identified in the macaques, with an average of 9.2 alleles per locus and an average heterozygosity of 0.76. Seven loci were analyzed using radiolabelled PCR primers and standard gel electrophoresis. Twelve loci were studied using fluorescently labelled primers and the Perkin-Elmer ABI 377 genotyping system. Of these 19 pigtailed macaque polymorphisms, 12 were used to perform paternity testing among captive animals. In a set of 15 infants, this panel of 12 genetic polymorphisms was sufficient to establish paternity in all cases. The number of alleles per locus in pigtailed macaques was compared with the number of alleles in a sample of baboons, and no significant correlation was observed. This indicates that population genetic processes such as genetic drift and recurrent mutation act rapidly enough on these loci to eliminate any relationship in levels of polymorphism across those two species. These 19 loci will be valuable for a range of genetic studies in pigtailed macaques, including paternity testing, analysis of population structure and differentiation among wild populations, and genetic linkage mapping.     

Isolation and characterization of highly polymorphic microsatellite loci in a pelagic‐spawning reef fish Hypodytes rubripinnis

Group spawning (1 female and more than 2 males) and streaking behaviour of subordinate males are common in pelagic‐spawning reef fishes such as the temperate tetrarogidid fish Hypodytes rubripinnis. Five microsatellite loci were isolated and characterized for paternity analysis of this species. Four of the five loci were highly polymorphic, having 11–34 alleles with observed heterozygosities between 0.89 and 1.00 in 37 unrelated individuals. These four loci conformed to Hardy–Weinberg equilibrium. These loci should be useful in future studies that assess how fertilization is partitioned among male H. rubripinnis in multimale spawning events.     

Development and characterization of novel microsatellite markers for the common earthworm (Lumbricus terrestris L.)

We developed and characterized 10 highly polymorphic microsatellite loci from an SSR‐enriched genomic DNA library of the common earthworm (Lumbricus terrestris L). Characterization of these loci using 32 individuals revealed high levels of genetic diversity, five to 18 alleles per locus and a high observed and expected heterozygosity. These loci will be used for paternity analysis and population genetic studies of the co‐evolution between L. terrestris and its parasites.     

Isolation and characterization of 11 polymorphic microsatellite loci in the giant white‐tailed rat (Uromys caudimaculatus)

Microsatellite loci were developed for the giant white‐tailed rat (Uromys caudimaculatus) to aid in assigning paternity and to subsequently investigate their mating system and sex‐biased dispersal characteristics. Twenty‐two primer sets were originally developed and of these, 11 were polymorphic with between five and 10 alleles per locus. In addition, two primer sets designed for Hydromys chyrsogaster also amplified in this species despite an evolutionary divergence of 15 million years (Myr).     

A molecular analysis of African lion (Panthera leo) mating structure and extra‐group paternity in Etosha National Park

The recent incorporation of molecular methods into analyses of social and mating systems has provided evidence that mating patterns often differ from those predicted by group social organization. Based on field studies and paternity analyses at a limited number of sites, African lions are predicted to exhibit a strict within‐pride mating system. Extra‐group paternity has not been previously reported in African lions; however, observations of extra‐group associations among lions inhabiting Etosha National Park in Namibia suggest deviation from the predicted within‐pride mating pattern. We analysed variation in 14 microsatellite loci in a population of 164 African lions in Etosha National Park. Genetic analysis was coupled with demographic and observational data to examine pride structure, relatedness and extra‐group paternity (EGP). EGP was found to occur in 57% of prides where paternity was analysed (n = 7), and the overall rate of EGP in this population was 41% (n = 34). Group sex ratio had a significant effect on the occurrence of EGP (P < 0.05), indicating that variation in pride‐level social structure may explain intergroup variation in EGP. Prides with a lower male‐to‐female ratio were significantly more likely to experience EGP in this population. The results of this study challenge the current models of African lion mating systems and provide evidence that social structure may not reflect breeding structure in some social mammals.     

Isolation and characterization of novel microsatellite loci for parentage assessment in the lance‐tailed manakin (Chiroxiphia lanceolata)

The lance‐tailed manakin (Chiroxiphia lanceolata) is a lek‐breeding bird from Central America in which males court females with complex cooperative displays. To resolve detailed patterns of paternity in the wild, we isolated and characterized 12 novel microsatellite loci in this species. Eleven of these loci were polymorphic (five to 14 alleles), with observed heterozygosities ranging from 0.36 to 0.87 (N = 574 individuals). We tested for linkage disequilibrium using randomized subsamples of adults to control for known family structure among long‐lived and sedentary individuals. These loci will be valuable in resolving paternity among many candidate fathers in this species.     

Assessment of multiple paternity in single litters from three species of carcharhinid sharks in Hawaii

We tested for presence or absence of multiple paternity in single litters from each of three congeneric shark species in Hawaii: the sandbar shark, Carcharhinus plumbeus, bignose shark, Carcharhinus altimus, and Galapagos shark, Carcharhinus galapagensis. Based on eight polymorphic microsatellite loci, we excluded paternity by a single sire in sandbar and bignose sharks, but could not exclude a single sire for the litter from the Galapagos shark. This study doubles the number of shark species tested for multiple paternity, and is the first demonstration of multiple paternity in sandbar and bignose sharks.     

Characterization of microsatellite loci isolated in midget faded rattlesnake (Crotalus viridis concolor)

Primers for five polymorphic microsatellite loci were developed for the midget faded rattlesnake (Crotalus viridis concolor), a rare subspecies of western rattlesnake (Crotalus viridus) found only in parts of Wyoming, Colorado, and Utah. Five polymorphic microsatellites were isolated, four of which had relatively high levels of diversity (eight to nine alleles). We found only two departures from Hardy–Weinberg equilibrium and they occurred in different loci, so null alleles are likely not a problem. Moreover, we found that no two loci were linked. These loci will be applicable for population genetic analysis and perhaps analysis of paternity and mating systems.     

Microsatellite loci for the Australian field cricket Teleogryllus oceanicus and their cross‐utility in Teleogryllus commodus

Microsatellite loci were isolated from the Australian field cricket Teleogryllus oceanicus. Seven polymorphic loci were found with an observed number of alleles ranging from eight to 17 and observed heterozygosities between 0.26 and 0.94. One locus was found to be X‐linked. These seven loci were also tested for amplification and polymorphism in the congeneric species Teleogryllus commodus. The loci will be used for paternity studies in these species.     

Ten polymorphic microsatellite DNA loci for paternity and population genetics analysis in the fen raft spider (Dolomedes plantarius)

Ten polymorphic di‐ and trinucleotide microsatellite loci were developed in the fen raft spider Dolomedes plantarius from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.62 to 0.9, with the observed allele numbers varying from four to 15 in the 22 individuals tested. Average paternity exclusion probabilities ranged between 0.290 and 0.686. In combination, the 10 polymorphic loci elicit an exclusion probability of 0.999. The high level of polymorphism of these microsatellite loci makes them ideal genetic markers for paternity and population genetics analysis in this endangered species.     

Microsatellite loci for the field cricket,Gryllus bimaculatus and their cross‐utility in other species of Orthoptera

We have isolated 16 microsatellite loci in the field cricket, Gryllus bimaculatus. Nine loci were found to be polymorphic in G. bimaculatus and the number of alleles varied from seven to 14. All 16 loci were tested for amplification in nine other species. In the five species tested belonging to the same subfamily (Gryllinae), a minimum of nine loci amplified. These loci will be used to determine paternity as part of a study to investigate the genetic benefits of polyandry.     

Isolation and characterization of microsatellite markers in musk duck (Biziura lobata: Aves), and their application to other waterfowl species

We isolated and characterized 11 novel microsatellite loci to study paternity in the Australian musk duck (Biziura lobata), using nonradioactive PCR‐based techniques to screen GA and GAAA repeats enriched genomic DNA libraries. Nine of 11 loci showed no evidence of null alleles and were variable (mean H_E = 0.825, mean number of alleles = 9). This set of nine loci is suitable for paternity assignment (exclusion probability for nine unlinked loci = 0.9999). We also demonstrated that many of these loci cross‐amplify in various other waterfowl species.     

Dispersal ability and invasion success of Crepidula fornicata in a single gulf: insights from genetic markers and larval-dispersal model

The success of an exotic species relies on many factors including dispersal capabilities and adaptation to novel environments. In particular, rapid spread from an initial point of introduction favours long-term establishment of exotic species, especially when large genetic diversity is maintained during the colonization phase. We here focused on the slipper limpet, Crepidula fornicata, a species native to the western Atlantic that has successfully invaded European bays and estuaries since the end of the nineteenth century following repeated introductions. Its settlement at high densities has major consequences on the macro-benthic fauna and flora. The aim of the present study was to analyse the ability of C. fornicata for rapid diffusion and long-distance dispersal, at the level of a large French gulf, namely the gulf of St-Malo (covering 120 km in latitude and 40 km in longitude) in the English Channel. The genetic architecture of 16 populations distributed all over this gulf was investigated using five microsatellite loci. Genetic diversity was found to be high and did not vary significantly with population density, population age or geographic location. Moreover, despite potential isolation among populations due to a strong tidal regime and the action of wind-induced currents, only weak barriers to gene flow were found across the gulf. These results were in agreement with results obtained from a simple 2D larval dispersal model. Both genetic data and the simulation model highlighted the potential for rapid and efficient spread of C. fornicata at a regional level.     

Isolation and characterization of polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum)

We report on the development of nine polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum) using a combination of enriched and unenriched subgenomic libraries. Based on the small percentage of positive clones in the unenriched library (0.4%) it appears that microsatellites are very scarce in nurse shark genomes. Numbers of alleles at polymorphic loci ranged from two to 15; observed heterozygosity ranged from 0.17 to 0.90. We expect these loci to be useful for studies of breeding structure and paternity.     

Isolation and characterization of microsatellite loci in Greater Sage‐Grouse (Centrocercus urophasianus)

Primers for five polymorphic microsatellite loci were developed for Greater Sage‐Grouse (Centrocercus urophasianus) using an enrichment/detection protocol. The high level of polymorphism (nine to 33 alleles) suggests that these loci will be applicable for investigating mating systems and paternity analysis as well as population genetics. Cross‐species amplification was successful for each locus in at least two other galliform species.