Strong population structure despite evidence of recent migration in a selfing hermaphroditic vertebrate, the mangrove killifish (Kryptolebias marmoratus)

We employ a battery of 33 polymorphic microsatellite loci to describe geographical population structure of the mangrove killifish (Kryptolebias marmoratus), the only vertebrate species known to have a mixed-mating system of selfing and outcrossing. Significant population genetic structure was detected at spatial scales ranging from tens to hundreds of kilometres in Florida, Belize, and the Bahamas. The wealth of genotypic information, coupled with the highly inbred nature of most killifish lineages due to predominant selfing, also permitted treatments of individual fish as units of analysis. Genetic clustering algorithms, neighbour-joining trees, factorial correspondence, and related methods all earmarked particular killifish specimens as products of recent outcross events that could often be provisionally linked to specific migration events. Although mutation is the ultimate source of genetic diversity in K. marmoratus, our data indicate that interlocality dispersal and outcross-mediated genetic recombination (and probably genetic drift also) play key proximate roles in the local 'clonal' dynamics of this species.     

Genetic variation for outcrossing among Caenorhabditis elegans isolates

The evolution of breeding systems results from the existence of genetic variation and selective forces favoring different outcrossing rates. In this study we determine the extent of genetic variation for characters directly related to outcrossing, such as male frequency, male mating ability, and male reproductive success, in several wild isolates of the nematode Caenorhabditis elegans. This species is characterized by an androdioecious breeding system in which males occur with hermaphrodites that can either self-fertilize or outcross with males. We find genetic variation for all characters measured, but also find that environmental variation is a large fraction of the total phenotypic variance. We further determine the existence of substantial genetic variation for population competitive performance in several laboratory environments. However, these measures are uncorrelated with outcrossing characters. The data presented here contribute to an understanding of male maintenance in natural populations through their role in outcrossing.     

Microsatellite documentation of male-mediated outcrossing between inbred laboratory strains of the self-fertilizing mangrove killifish (Kryptolebias Marmoratus)

Primers for 36 microsatellite loci were developed and employed to characterize genetic stocks and detect possible outcrossing between highly inbred laboratory strains of the self-fertilizing mangrove killifish, Kryptolebias marmoratus. From attempted crosses involving hermaphrodites from particular geographic strains and gonochoristic males from others, 2 among a total of 32 surveyed progenies (6.2%) displayed multilocus heterozygosity clearly indicative of interstrain gametic syngamy. One of these outcross hybrids was allowed to resume self-fertilization, and microsatellite assays of progeny showed that heterozygosity decreased by approximately 50% after one generation, as expected. Although populations of K. marmoratus consist mostly of synchronous hermaphrodites with efficient mechanisms of internal self-fertilization, these laboratory findings experimentally confirm that conspecific males can mediate occasional outcross events and that this process can release extensive genic heterozygosity.     

Environmental diel variation, parasite loads, and local population structuring of a mixed-mating mangrove fish

Genetic variation within populations depends on population size, spatial structuring, and environmental variation, but is also influenced by mating system. Mangroves are some of the most productive and threatened ecosystems on earth and harbor a large proportion of species with mixed-mating (self-fertilization and outcrossing). Understanding population structuring in mixed-mating species is critical for conserving and managing these complex ecosystems. Kryptolebias marmoratus is a unique mixed-mating vertebrate inhabiting mangrove swamps under highly variable tidal regimes and environmental conditions. We hypothesized that geographical isolation and ecological pressures influence outcrossing rates and genetic diversity, and ultimately determine the local population structuring of K. marmoratus. By comparing genetic variation at 32 microsatellites, diel fluctuations of environmental parameters, and parasite loads among four locations with different degrees of isolation, we found significant differences in genetic diversity and genotypic composition but little evidence of isolation by distance. Locations also differed in environmental diel fluctuation and parasite composition. Our results suggest that mating system, influenced by environmental instability and parasites, underpins local population structuring of K. marmoratus. More generally, we discuss how the conservation of selfing species inhabiting mangroves and other biodiversity hotspots may benefit from knowledge of mating strategies and population structuring at small spatial scales.     

POPULATION STRUCTURE AND ESTIMATES OF GENE FLOW IN THE HOMOSPOROUS FERN POLYSTICHUM MUNITUM

Levels and distribution of genetic variation were investigated in the homosporous fern, Polystichum munitum. Homosporous ferns differ from higher vascular plants in that they possess potentially bisexual gametophytes which can produce a completely homozygous sporophyte in a single generation. Because of this, it has long been maintained that ferns possess an inbreeding mating system, resulting in low levels of genetic variation and high levels of homozygosity within populations. The four populations sampled maintain high levels of genetic variation (P? = 0.542; H? = 0.111; ā = 2.23), comparable to that maintained by populations of outcrossing seed plants. The mean fixation index, F, for the four populations was 0.052, indicating no significant deviations from Hardy-Weinberg genotypic expectations. Polystichum munitum distributes most of its genetic variation within rather than among populations. Population-genetic structure was assessed by subdividing each of two large populations into 10 × 10-m subpopulations. Comparisons of genetic variation within and among subpopulations indicated little genetic substructure within either of the artificially subdivided populations. Estimates of interpopulational gene flow (Nm) are extremely high, comparable to those reported for gymnosperms. Statistical estimates of intragametophytic selling are very low, ranging from 0 to 3%. This study suggests that Polystichum munitum is an outcrossing species. Evidence from this and other investigations indicates that fern species do not typically self-fertilize and that mating systems in ferns vary as they do among species of seed plants.     

Gender-specific inbreeding depression in a gynodioecious plant, Geranium maculatum (Geraniaceae)

In gynodioecious species, females coexist with hermaphrodites in natural populations even though hermaphrodites attract more pollinators, are capable of reproducing through pollen, and can self-fertilize. This study tests the hypothesis that inbreeding depression helps to maintain females in natural populations. It also examines whether gender lineages that differ in selfing rates might experience different levels of inbreeding depression. Female and hermaphroditic lineages of the gynodioecious species Geranium maculatum were used in self, sib-cross and outcross experiments to examine inbreeding depression levels and to determine whether these levels differ between hermaphroditic and female lineages. Six fitness correlates were measured in the greenhouse and compared among pollination types and between genders. Severe inbreeding depression was found for both individual fitness traits and cumulative fitness in early life history stages. Inbreeding depression levels were slightly higher in hermaphroditic than in female lineages, but this difference was not statistically significant. Because females are unable to self-pollinate and are less likely to experience inbreeding than hermaphrodites under natural conditions, these results suggest that severe inbreeding depression could confer a selective advantage for females that could help to maintain females in natural populations.     

Does selfing or outcrossing promote local adaptation?

The degree to which plants self-fertilize may impact their potential for genetic adaptation. Given that the mating system influences genetic processes within and among populations, the mating system could limit or promote local adaptation. I conducted a literature survey of published reciprocal transplant experiments in plant populations to quantify the effect of mating system on the magnitude of local adaptation. Mating system had no effect on local adaptation. I detected no effect when species were categorized as either self-compatible or self-incompatible or when accounting for environmental differences between source populations. The results suggest that, despite limited genetic variation in selfing species and greater potential for gene flow in outcrossing species, mating system has little influence on adaptation of populations.     

Leaf litter decomposition differs among genotypes in a local <Emphasis Type="Italic">Betula pendula</Emphasis> population

Ecosystem processes, such as plant litter decomposition, are known to be partly genetically determined, but the magnitude of genetic variation within local populations is still poorly known. We used micropropagated field-grown saplings of 19 Betula pendula genotypes, representing genetic variation in a natural birch population, to examine (1) whether genotype can explain variation in leaf litter decomposition within a local plant population, and (2) whether genotypic variation in litter decomposition is associated with genotypic variation in other plant attributes. We found that a local B. pendula population can have substantial genotypic variation in leaf litter mass loss at the early stages of the decomposition process and that this variation can be associated with genotypic variation in herbivore resistance and leaf concentrations of soluble proteins and total nitrogen (N). Our results are among the first to show that fundamental ecosystem processes can be significantly affected by truly intraspecific genetic variation of a plant species.     

Genital dimorphism in natural populations of the land snail Chondrina clienta and the influence of the environment on its expression

Several species of simultaneously hermaphroditic land snails show a genital dimorphism: aphallic individuals differ from euphallic ones by a lack of male copulatory organs (penis plus genital retractor muscle). Aphallic individuals can self-fertilize or outcross as females but not as males. Thus, the mating system of a population may be significantly influenced by the proportion of aphallic individuals.
We present data on the frequency of aphally in 23 natural populations of the rock-dwelling land snail Chondrina clienta on the Baltic island of Öland, Sweden. The populations varied greatly in percentage of aphallic individuals, ranging from 52.2 to 99.1%, (grand mean 77.7%)). This variation did not follow any geographical pattern.
In a laboratory experiment, we examined whether food supply (high or low) and/or population density (high or low) experienced during ontogeny affected the expression of genital dimorphism. Snails derived from a population with 99.1% aphallic individuals and raised under different food and density conditions did not differ from the original population in frequency of aphally. By contrast, when snails from a population with 66.7% aphallic individuals were raised on a low food supply, more individuals became euphallic than expected under complete genetic determination. These results suggest that, in addition to a genetic component. the expression of the genital dimorphism can be influenced by environmental conditions.     

BEST OF BOTH WORLDS? ASSOCIATION BETWEEN OUTCROSSING AND PARASITE LOADS IN A SELFING FISH

Mixed-mating strategies (i.e., intermediate levels of self-fertilization and outcrossing in hermaphrodites) are relatively common in plants and animals, but why self-fertilization (selfing) rates vary so much in nature has proved difficult to explain. We tested the hypothesis that parasites help maintain mixed-mating using a partially selfing fish (Kryptolebias marmoratus) as a model. We show that outcrossed progeny in the wild are genetically more diverse and less susceptible to multiple parasite infections than their selfed counterparts. Given that outcrossing in K. marmoratus can only be attained by male-hermaphrodite matings, our data provide an explanation for the coexistence of males and hermaphrodites in androdioecious species where hermaphrodites are unable to outcross among themselves. Moreover, our study provides evidence that parasites contribute to maintaining mixed-mating in a natural animal population.     

The Importance of Plant Provenance and Genotypic Diversity of Seed Material Used for Ecological Restoration

The increased translocation of plant species for biodiversity restoration and habitat creation has provoked a debate on provenance and genotypic diversity of the used plant material. Nonlocal provenances are often not adapted to the local environmental conditions, and low population genotypic diversity may result in genetic bottlenecks hampering successful establishment. We tested provenance differentiation of four plant species used in agri‐environment schemes to increase biodiversity of agricultural landscapes (wildflower strips). Provenances were collected close to the experimental field and at four further sites of different distances ranging from 120 to 900 km. In two of these provenances, different levels of genotypic diversity were simulated by sowing seed from a high and low number of mother plants. We found a large provenance differentiation in fitness‐related traits, particularly in seedling emergence. There was no evidence for a general superiority of the local population. The productivity was greater in populations of high genotypic diversity than in those of low diversity, but the effect was only significant in one species. Productivity was also more constant among populations of high diversity, reducing the risk of establishment failure. Our results indicate that the choice of an appropriate provenance and a sufficient genotypic diversity are important issues in ecological restoration. The use of local provenances does not always guarantee the best performance, but a spread of superior alien genotypes can be avoided. A sufficient genotypic diversity of the sown plants might be a biological insurance against fluctuations in ecosystem processes increasing the reliability of restoration measures.     

Pollen discounting and the evolution of selfing in Arenaria uniflora (caryophyllaceae)

Although most models of mating system evolution assign a central role to the male transmission advantage of selfing genotypes, empirical data on the male fitness consequences of increased self-pollination are still uncommon. Here, I use measures of pollen import and export by focal plants in genotyped arrays to investigate the effects of floral morphology and pollination environment on self and outcross male function. Plants from an autogamous population of Arenaria uniflora (Caryophyllaceae) exhibit complete pollen discounting relative to closely related outcrossers, as do morphologically intermediate F1 hybrids between the two populations. However, the low cumulative male fitness of hybrids probably results from reduced pollen number or competitive ability, rather than a nonlinear relationship with floral morphology. When surrounded by selfers, plants from the outcrosser population self-fertilize at nearly the same rate as selfers (>80%), but have much lower self male fitness due to reduced fruit set. Because outcross siring success is also extremely low (<8%) in this treatment, these mate-limited outcrossers are at male fitness disadvantage to both pseudocleistogamous selfers and nonlimited outcrossers. The relative male fitness of plants with different mating systems appears dependent on the ecological context, as well as on morphological trade-offs.     

The expression of self-incompatibility in angiosperms is bimodal

Self-incompatibility is expressed by nearly one-half of all angiosperms. A large proportion of the remaining species are self-compatible, and they either outcross using various contrivances or self-fertilize to some extent. Because of the common occurrence of populations and individuals with intermediate levels of self-incompatibility, categorization of the expression of self-incompatibility as an approximately binary trait has become controversial. We collect a widely reported index (index of self-incompatibility [ISI]) used to asses the strength and variation of self-incompatibility from over 1200 angiosperm taxa. Its distribution is bimodal and positively associated with outcrossing rate, albeit with a weak relationship within self-compatible taxa. A substantial fraction of species has intermediate mean values of ISI. Their occurrence can be caused by segregating ephemeral self-compatible mutations, averaging artifacts, and experimental biases, in addition to the often invoked stabilizing selection acting on the expression of self-incompatibility. Selection may also generally favor taxa with high ISI values through increased lineage birth and death rates, and it may counter lower level selection advantages within taxa expressing intermediate and low values of ISI. Such a null hypothesis is nearly universally overlooked, despite the fact that it could adequately explain the observed distribution of mating and breeding systems.     

Epigenetic regulation of sex ratios may explain natural variation in self-fertilization rates

Self-fertilization (selfing) favours reproductive success when mate availability is low, but renders populations more vulnerable to environmental change by reducing genetic variability. A mixed-breeding strategy (alternating selfing and outcrossing) may allow species to balance these needs, but requires a system for regulating sexual identity. We explored the role of DNA methylation as a regulatory system for sex-ratio modulation in the mixed-mating fish Kryptolebias marmoratus. We found a significant interaction between sexual identity (male or hermaphrodite), temperature and methylation patterns when two selfing lines were exposed to different temperatures during development. We also identified several genes differentially methylated in males and hermaphrodites that represent candidates for the temperature-mediated sex regulation in K. marmoratus. We conclude that an epigenetic mechanism regulated by temperature modulates sexual identity in this selfing species, providing a potentially widespread mechanism by which environmental change may influence selfing rates. We also suggest that K. marmoratus, with naturally inbred populations, represents a good vertebrate model for epigenetic studies.     

Newts under siege: range expansion of Triturus pygmaeus isolates populations of its sister species

The newt species Triturus marmoratus and Triturus pygmaeus are both present in central Portugal where they have parapatric distributions. We used four genetic markers to determine which species was present in 31 populations. In the centre of the study area we found a T. marmoratus enclave. Despite small interpopulation distances, hybridization is locally rare. We built several models to try to explain this distribution using environmental data. The best model, chosen by Akaike's Information Criterion, relates the presence of T. marmoratus with the temperature in July, the relief of the landscape, and a higher use of the land for orchards. The current distribution can best be explained by T. pygmaeus expanding north and replacing T. marmoratus , the latter only persisting where ecological conditions are relatively favourable.     

Genetic differentiation of the millipede Pycnotropis epiclysmus inhabiting seasonally inundated and non-flooded Amazonian forests

The millipede Pycnotropis epiclysmus Hoffman, 1995 (Diplopoda: Polydesmida: Platyrhacidae) is frequently found in Central Amazonian white-and mixed-water inundation forests along the Solimöes-Amazon River near Manaus, Brazil. It also inhabits non-flooded disturbed forest areas adjacent to this river. Populations from both biotopes were genetically studied. The specific pPeP172 satellite DNA family identified in P. epiclysmus has been analyzed in order to elucidate the systematic rank of morphologically indistinguishable individuals from the different habitat types. Nucleotide sequence data, sequence variability and copy number estimates of the pPeP172 satellite DNA do not discriminate the respective populations into genetically different ecotypes. The study of enzyme variability, however, revealed genotypic differences among the three populations: the populations from two geographically more distant inundation forests are genetically rather similar; and the geographically closer populations, one found in a non-flooded and the other in an inundation forest, have a genetic distance which is of similar magnitude to that of the two geographically more distant populations. The genetic data suggest that individuals from different habitats belong to populations of a single species. Genotypic structuring among and within local populations indicates processes of genetic differentiation which can be the result of the migration ability of this millipede.     

Phylogenetic relationships and introgression patterns between incipient parapatric species of Italian brown trout (Salmo trutta L. complex)

Genetic variation at 47 protein loci was investigated in 16 wild brown trout populations from the Pô basin and three major domesticated stocks used for stocking this area. Twenty-four loci were polymorphic and large frequency differences were found at 15 of them. The most significant allozyme variations were congruent with the mtDNA sequence polymorphism previously observed in the same samples. We confirmed the occurrence of two parapatric incipient species, Salmo marmoratus and S. trutta fario , previously identified by morphological traits. These two species were fixed or nearly fixed for alternate alleles at eight loci (Nei's standard genetic distance = 0.16–0.18), but introgression was detected between adjacent samples of the two forms. Divergence levels at both mtDNA and nuclear loci suggested that the differentiation between S. marmoratus and S. trutta fario started between 3 and 1 million years before present. Variation at protein loci and mtDNA supported the hypothesis that the third species found in this area, S. carpio (an endemic population of the lake Garda) was issued from a recent hybridization of the two first species. Finally, we showed that three of the major Italian fish-farm strains originated from the Atlantic side and displayed substantial genetic differences with the natural populations of the Pô basin. Most of these populations were contaminated by stocking with introgression rate ranging from 0 to 70% and measures of protection and restoration of the rich genetic diversity present in this area should be urgently applied.     

Modulatory effect of environmental endocrine disruptors on N-ras oncogene expression in the hermaphroditic fish, Kryptolebias marmoratus

Kryptolebias marmoratus is the only known internally self-fertilizing vertebrate. It shows high susceptibility to many chemical carcinogens and has been proposed as a potential cancer model species alternative to mammals. Since use of this fish species is expected to rise in cancer research, regulation of oncogenes from K. marmoratus needs proper understanding. We cloned and deduced full-length sequence of cDNA of N-ras oncogene from K. marmoratus. Study of expression profile of N-ras by using quantitative real-time RT-PCR revealed that brain had the highest level of expression compared to other tissues. Some embryonic stages showed more N-ras expression than juveniles and adults. Exposure to two environmental endocrine disrupting chemicals (EDCs), bisphenol A (BPA) and 4-nonylphenyl (NP) caused up-regulation of N-ras in gonad, intestine and liver of hermaphrodite K. marmoratus. It is suggested that K. marmoratus may be a suitable model species for oncogene expression studies. The observed EDC-induced expression of N-ras supports the assumption that EDC exposure may predispose the host to the risk of environmental carcinogenesis.     

THE GENETIC STRUCTURE OF LARGE-LAKE DAPHNIA POPULATIONS

Cyclical parthenogenesis allows study of the genetic and evolutionary characteristics of groups exhibiting both asexual and sexual reproduction. The cladoceran genus Daphnia contains species which vary with respect to the relative incidence of sexual reproduction; pond species tend to undergo sexual reproduction more regularly than species found in large lakes. Previous genetic studies have focused on pond populations, generating expectations about large-lake populations that have not been fully met by recent studies. The present study of the Palearctic species Daphnia galeata further examines the genetic structure of large-lake populations. Nine local populations, from lakes in northern Germany, are examined for genetic variation at seven enzyme loci. Populations exhibit similar allelic arrays and often similar allele frequencies at the five polymorphic loci; values of Nei's genetic distance (D) ranged from 0.002 to 0.239, with a mean of 0.084. F_ST values range from 0.012 to 0.257, and spatial autocorrelation coefficients range from -0.533 to 0.551, for the eight alleles analyzed. With few exceptions, within-population genotypic frequencies were in Hardy-Weinberg equilibrium. There was, however, significant heterogeneity in genotypic frequencies among populations. The number of coexisting clonal groups, as determined by three locus genotypes, is high within populations. Clonal groups are widely distributed among localities. The amount of genetic divergence observed among these large-lake populations is smaller than that previously observed among pond populations and suggests that different processes may be important in determining the genetic structure and subsequent phenotypic divergence of lake versus pond populations.