Dynamic management of genetic resources: maintenance of outcrossing in experimental metapopulations of a predominantly inbreeding species

Dynamic management of genetic resources inpredominantly inbreeding species requiresincreased levels of outcrossing to limit theloss of genetic variation due to smallereffective sizes and to favour the emergence ofnew genetic combinations. Here, we show thatoutcrossing rates can artificially bepermanently increased in experimental evolvingplant metapopulations, using Arabidopsisthaliana as a model. We introducedmale-sterility genes and used an adequatemanagement of the resulting female plants tomodify the outcrossing rates. As expected, theincrease in outcrossing resulted in lowerlevels of heterozygote deficiency (F _is) than observed in natural populationsof A. thaliana and therefore in themaintenance of potentially higher levels ofgenetic variation. An additional selectiveadvantage for females' offspring, due to theproduction of larger seeds by females and apossible heterosis effect, furthermore led tosmaller F _is than expected from therealized outcrossing rate. This selectiveadvantage also resulted in an increase infemale frequency, especially in metapopulationswith large population sizes, creating anon-causal negative correlation between femalefrequency and heterozygote deficiency.     

OneMap: software for genetic mapping in outcrossing species

OneMap is an environment for constructing linkage maps of outcrossing plant species, using full-sib families derived from two outbred parents. The analyses are performed using a novel methodology based on the maximum likelihood approach for simultaneous estimation of linkage and linkage phases (Wu et al. 2002), which has been successfully applied to sugarcane (Garcia et al. 2006). It is implemented as a set of functions for the freely distributed software R, and handles pairwise marker analysis, marker ordering and map refinement. The software is freely available at http://www.ciagri.usp.br/ approximately aafgarci/OneMap/.     

Variation in outcrossing rate and population genetic structure of Clarkia tembloriensis (Onagraceae)

Summary Outcrossing rate estimates for eight accessions of Clarkia tembloriensis indicate that this annual plant species has a wide interpopulational range of outcrossing rate ( ). Populations' t estimates were significantly correlated with observed heterozygosity and mean number of alleles per locus. Estimated fixation indices, , for most populations were very close to their expected values, F_eq, for a given Nei's gene diversity statistics showed that the group of outcrossing populations have more total genetic variation and less differentiation among populations than does the group of selfing populations. These results indicate that the breeding system of C. tembloriensis has had a strong influence on the amount and distribution of genetic variation within and among its populations.     

Effects of epistasis on tests for linkage in self-pollinated species

Summary Tests for linkage based on covariances among relatives in self-pollinated species are usually based upon an assumption that epistasis is not important. This study was conducted to determine the impact of epistasis on, and to investigate the sensitivity of, such tests. Thirty covariances were calculated for each of ten non-epistatic and ten epistatic genetic models with varying probabilities of recombination between two coupling or repulsion loci. Each set of covariances was tested for linkage by comparing covariances calculated for the model with those expected for an additive-dominance model with no linkage. Results showed that the test for linkage is quite insensitive to the effects of linkage due to the disproportionate influence of inbreeding. Repulsion linkages should be easier to detect than coupling linkages for all models. Epistasis was found to mimic or counteract the effects of linkage. Tests for linkage based on covariances within a hierarchical mating design appear to be insensitive to linkage and may confuse the effects of linkage and epistasis.     

Comparison of three genetic similarity coefficients based on dominant markers from predominantly self-pollinating species

Three genetic similarity coefficients were estimated and compared for their usefulness: simple matching (S _SM), Jaccard’s (S _J) and Dice’s (S _D), all based on dominant markers data from individuals representing predominantly self-pollinating species. AFLP markers were used to analyze 139 Phaseolus vulgaris L. (common bean) and 67 Lactuca saligna L. (least lettuce) accessions, and RAPD markers were used to analyze 110 Triticum dicoccoides Koern. (wild emmer wheat) accessions. Similar discriminating structure and power based on the three genetic similarity coefficients was found for each of the three species. This discriminating power was high for both P. vulgaris and L. saligna but moderate for T. dicoccoides. With closely related individuals, as in our study, the absence of a band in two individuals should be due to an identical cause inherited from the same ancestor. Accordingly we propose the use of S _SM, which alone out of the three examined coefficients involved shared absence of DNA bands, as contributing to genetic similarity. When RAPDs are employed, inferences about population structure and nucleotide divergence should be made with prudence as the nature of genetic variation uncovered by RAPDs is often unclear.     

Comparison of the fine-scale genetic structure of three dipterocarp species

We investigated the fine-scale genetic structure of three tropical-rainforest trees, Hopea dryobalanoides, Shorea parvifolia and S. acuminata (Dipterocarpaceae), in Peninsular Malaysia, all of which cooccurred within a 6-ha plot in Pasoh Forest Reserve. A significant genetic structure was found in H. dryobalanoides, weaker (but still significant) genetic structure in S. parvifolia and nonsignificant structure in S. acuminata. Seeds of all three species are wind dispersed, and their flowers are thought to be insect pollinated. The most obvious difference among these species is their height: S. parvifolia and S. acuminata are canopy species, whereas H. dryobalanoides is a subcanopy species. Clear differences were also found among these species in their range of seed dispersal, which depends on the height of the release point; so taller trees disperse their seed more extensively. The estimates of seed dispersal area were consistent with the degree of genetic structure found in the three species. Therefore, tree height probably had a strong influence on the fine-scale genetic structure of the three species.     

Internal genetic structure and outcrossing rate in a natural population of Araucaria angustifolia (Bert.) O. Kuntze

The internal genetic structure and outcrossing rate of a population of Araucaria angustifolia (Bert.) O. Kuntze were investigated using 16 allozyme loci. Estimates of the mean number of alleles per loci (1.6), percentage of polymorphic loci (43.8%), and expected genetic diversity (0.170) were similar to those obtained for other gymnosperms. The analysis of spatial autocorrelation demonstrated the presence of internal structure in the first distance classes (up to 70 m), suggesting the presence of family structure. The outcrossing rate was high (0.956), as expected for a dioecious species. However, it was different from unity, indicating outcrossings between related individuals and corroborating the presence of internal genetic structure. The results of this study have implications for the methodologies used in conservation collections and for the use or analysis of this forest species.     

Patterns of genetic diversity in outcrossing and selfing populations of Arabidopsis lyrata

Arabidopsis lyrata is normally considered an obligately outcrossing species with a strong self-incompatibility system, but a shift in mating system towards inbreeding has been found in some North American populations (subspecies A. lyrata ssp. lyrata). This study provides a survey of the Great Lakes region of Canada to determine the extent of this mating system variation and how outcrossing rates are related to current population density, geographical distribution, and genetic diversity. Based on variation at microsatellite markers (progeny arrays to estimate multilocus outcrossing rates and population samples to estimate diversity measures) and controlled greenhouse pollinations, populations can be divided into two groups: (i) group A, consisting of individuals capable of setting selfed seed (including autogamous fruit set in the absence of pollinators), showing depressed outcrossing rates (T(m) = 0.2-0.6), heterozygosity (H(O) = 0.02-0.06) and genetic diversity (H(E) = 0.08-0.10); and (ii) group B, consisting of individuals that are predominantly self-incompatible (T(m) > 0.8), require pollinators for seeds set, and showing higher levels of heterozygosity (H(O) = 0.13-0.31) and diversity (H(E) = 0.19-0.410). Current population density is not related to the shift in mating system but does vary with latitude. Restricted gene flow among populations was evident among all but two populations (F(ST) = 0.11-0.8). Group A populations were more differentiated from one another (F(ST) = 0.78) than they were from group B populations (F(ST) = 0.59), with 41% of the variation partitioned within populations, 47% between populations, and 12% between groups. No significant relationship was found between genetic and geographical distance. Results are discussed in the context of possible postglacial expansion scenarios in relation to loss of self-incompatibility.     

High outcrossing in the annual colonizing species Ambrosia artemisiifolia (Asteraceae)

Background and Aims

Variation in mating patterns may be particularly evident in colonizing species because they commonly experience wide variation in plant density. Here, the role of density for the mating system of Ambrosia artemisiifolia (common ragweed), a wind-pollinated annual colonizing species previously reported as self-compatible, is explored.

Methods

The effect of population density on the proportion of self- and cross-fertilized seeds was examined using allozyme markers and experimental arrays conducted over two seasons in the field. Also the reproductive success of isolated plants located in diverse habitats was measured. The potential occurrence of a physiological mechanism preventing self-fertilization, i.e. self-incompatibility, following controlled self- and cross-pollinations in the glasshouse was examined.

Key Results

Outcrossing rates estimated using allozyme markers were uniformly high, regardless of the spacing between plants. However, when single plants were isolated from congeners they set few seeds. Observations of pollen-tube growth and seed set following controlled pollinations demonstrated that plants of A. artemisiifolia possess a strong self-incompatibility mechanism, contrary to earlier reports and assumptions.

Conclusions

The maintenance of high outcrossing rates in colonizing populations of A. artemisiifolia is likely to be facilitated by the prodigious production of wind-borne pollen, high seed production and extended seed dormancy.Key words: Self-incompatibility, outcrossing rate, density dependence, colonization, wind-pollination, Ambrosia artemisiifolia (ragweed), Asteraceae     

Simultaneous maximum likelihood estimation of linkage and linkage phases in outcrossing species

With the advent of new molecular marker technologies, it is now feasible to initiate genome projects for outcrossing plant species, which have not received much attention in genetic research, despite their great agricultural and environmental value. Because outcrossing species typically have heterogeneous genomes, data structure for molecular markers representing an entire genome is complex: some markers may have more alleles than others, some markers are codominant whereas others are dominant, and some markers are heterozygous in one parent but fixed in the other parent whereas the opposite can be true for other markers. A major difficulty in analyzing these different types of marker at the same time arises from uncertainty about parental linkage phases over markers. In this paper, we present a general maximum-likelihood-based algorithm for simultaneously estimating linkage and linkage phases for a mixed set of different marker types containing fully informative markers (segregating 1:1:1:1) and partially informative markers (or missing markers, segregating 1:2:1, 3:1, and 1:1) in a full-sib family derived from two outbred parent plants. The characterization of linkage phases is based on the posterior probability distribution of the assignment of alternative alleles at given markers to two homologous chromosomes of each parent, conditional on the observed phenotypes of the markers. Two- and multi-point analyses are performed to estimate the recombination fraction and determine the most likely linkage phase between different types of markers. A numerical example is presented to demonstrate the statistical properties of the model for characterizing the linkage phase between markers.     

Phylogeography of the crown-of-thorns starfish: genetic structure within the Pacific species

Previous studies of the population genetic structure of the corallivorous crown-of-thorns starfish (COTS) Acanthaster planci in the Pacific Ocean showed high levels of gene flow that were assumed to reflect a high dispersal potential. However, the phylogeographic analyses of the Pacific crown-of-thorns starfish species of this study, using the highly variable mitochondrial control region and the most complete geographic coverage to date, contradict this view. Results show high levels of overall genetic structure (Φ_ST = 0.198), suggesting a complex history of range restrictions and expansions, a pattern that we hypothesize results from changes in topography and oceanography associated with sea-level changes. However, results also show signatures of ongoing gene flow between populations isolated in the past and high levels of genetic connectivity even among distant populations. Combined, these results indicate that while there are significant limits to genetic exchange among populations among Pacific Ocean populations of the crown-of-thorns starfish, the high larval dispersal potential of this species is often achieved as well.     

Balance between inbreeding and outcrossing in a nannandrous species,the moss Homalothecium lutescens

Epiphytic dwarf males on the females present a possible solution to the problem of short fertilization distances in mosses. However, leptokurtic spore dispersal makes dwarf males likely to be closely related to their host shoot, with an accompanying risk of inbreeding. The capacity of a female to harbour a high number of different dwarf males suggests that there may be mechanisms in place that counteract inbreeding, such as polyandry and post-fertilization selection. We have genotyped sporophytes, female host shoots and dwarf males in four populations of the moss Homalothecium lutescens. We found no evidence of selective sporophyte abortion based on level of heterozygosity. The occurrence of entirely homozygous sporophytes together with significantly positive inbreeding coefficients in three of the populations (mean F_IS between 0.48 and 0.64) suggest frequent mother–son mating events. However, 23% of all sampled sporophytes had a higher level of heterozygosity compared with the mean expected heterozygosity at the population level. Polyandry was frequent, on average 59% of the sporophytes on a female shoot were sired by distinct fathers. In conclusion, sporadic fertilizations by dwarf males originating from nonhost female shoots appear to counteract strong inbreeding.     

Population genetic structure of two water strider species in the Ecuadorian Amazon

1. We used mtDNA sequence variation to estimate population genetic structure between and among water strider populations of Potamobates williamsi and P . sumaco in the Ecuadorian Amazon.
2. Sequencing of the COI mitochondrial gene revealed 16 haplotypes, which were summarised into four haplotype groups. P . williamsi and P . sumaco shared two common and widespread haplotypes.
3. Population structure was moderate and gene flow was low. Both air and river distances were significantly correlated with gene flow and, thus, indicative of isolation by distance.
4. The genetic structure within and among populations of P. williamsi and P. sumaco was probably not influenced by the dynamic tropical lotic system.     

Identifying genetic components controlling fertility in the outcrossing grass species perennial ryegrass (Lolium perenne) by quantitative trait loci analysis and comparative genetics

Mutational load and resource allocation factors and their effects on limiting seed set were investigated in ryegrass by comparative mapping genomics and quantitative trait loci (QTL) analysis in two perennial ryegrass (Lolium perenne) mapping families sharing common genetic markers. Quantitative trait loci for seed-set were identified on chromosome (LG) 7 in both families and on LG4 of the F2/WSC family. On LG7, seed-set and heading date QTLs colocalized in both families and cannot be unequivocally resolved. Comparative genomics suggests that the LG7 region is syntenous to a region of rice LG6 which contains both fertility (S5(n)) and heading date (Hd1, Hd3a) candidate genes. The LG4 region is syntenous to a region of rice LG3 which contains a fertility (S33) candidate gene. QTL maxima for seed-set and heading date on LG4 in the F2/WSC family are separated by c. 8 cm, indicating distinct genetic control. Low seed set is under the control of recessive genes at both LG4 and LG7 locations. The identification of QTLs associated with seed set, a major component of seed yield in perennial ryegrass, indicates that mutational load associated with these genomic regions can be mitigated through marker-assisted selection.     

Population structure and genetic diversity in two species of Hawaiian picture-winged Drosophila

Over the last several decades many picture-winged Drosophila have become less common in both geographical distribution and local population size (pers. obs., Foote pers. comm., Montgomerey pers. comm.). Here we report on a study of two Hawaiian Drosophila species, D. engyochracea, and D. hawaiiensis, to determine the impact that changes in population sizes over the past thirty years have had on the genetic diversity of these species. D. engyochracea is known from only two locations on the Island of Hawai'i (Kipuka Ki and Kipuka Pua'ulu), while D. hawaiiensis is currently more wide spread across Hawai'i Island. We collected 65 D. hawaiiensis and 66 D. engyochracea from two forest patches (kipuka) isolated by a 400 year old volcanic ash deposit. DNA sequence data for 515 bases of the mitochondrial gene COII was analyzed for both species to estimate relative total genetic diversity as well as inter-kipuka gene flow. The more wide spread species, D. hawaiiensis, has more genetic diversity (23 vs. 11 unique haplotypes) than the rarer species, D. engyochracea. The distribution of haplotypes in the kipuka is consistent with more gene flow in D. engyochracea than in D. hawaiiensis. Phylogenetic analysis indicates a small number of individuals morphologically identified as one species but have DNA sequence diagnostic for the other species. These results are consistent with these individuals being descendant from hybrids between species.     

Different levels of inbreeding depression between outcrossing and selfing Serapias species

We quantified inbreeding depression for fruit production, embryo vitality and seed germination in three deceptive orchids, Serapias vomeracea, S. cordigera and S. parviflora, which do not provide any reward to their pollinators, and are predicted to experience high outcrossing. Of the three species examined only S. parviflora was autonomously selfing. Both S. vomeracea and S. cordigera showed highly significant differences in fitness between selfed and outcrossed progenies, resulting in high levels of inbreeding depression, which increased in magnitude from seed set to seed germination. Inbreeding depression may promote outcrossing in Serapias by acting as a post-pollination barrier to selfing. Cumulative inbreeding depression across three stages in S. parviflora was lower that in both outcrossing species. The large difference in germination between selfed and outcrossed seeds is an important issue in conservation biology.     

High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations

BACKGROUND: Caenorhabditis elegans is a major model system in biology, yet very little is known about its biology outside the laboratory. In particular, its unusual mode of reproduction with self-fertile hermaphrodites and facultative males raises the question of its frequency of outcrossing in natural populations. RESULTS: We describe the first analysis of C. elegans individuals sampled directly from natural populations. C. elegans is found predominantly in the dauer stage and with a very low frequency of males versus hermaphrodites. Whereas C. elegans was previously shown to display a low worldwide genetic diversity, we find by comparison a surprisingly high local genetic diversity of C. elegans populations; this local diversity is contributed in great part by immigration of new alleles rather than by mutation. Our results on heterozygote frequency, male frequency, and linkage disequilibrium furthermore show that selfing is the predominant mode of reproduction in C. elegans natural populations but that infrequent outcrossing events occur, at a rate of approximately 1%. CONCLUSIONS: Our results give a first insight in the biology of C. elegans in the natural populations. They demonstrate that local populations of C. elegans are genetically diverse and that a low frequency of outcrossing allows for the recombination of these locally diverse genotypes.     

Fine-scale genetic structure, phylogeny and systematics of threatened crayfish species complex

Systematic uncertainties in the crayfish Austropotamobius pallipes are well grounded by the number of species and subspecies described using different approaches, causing scientists to define this taxon as "complex". However, a key task that conservation programmes are facing regarding the recent and drastic decline of European populations, is the coherent systematic classification of this threatened species. Here we present results obtained by coupling mtDNA and genome analysis suggestive of a novel evolutionary framework to explain the relationships among phylogenetic lineages of A. pallipes. The direct sequencing of mtDNA COI gene fragment revealed a strong geographic structure with four distinct haplogroups separated by a range of 5-25 mutations. However, mitochondrial data were not supported by genomic fingerprinting based on 535 AFLP polymorphisms. Nuclear markers showed an unexpected moderate level of genetic differentiation and the absence of any geographic structure. Consequently, this study proposes that the taxonomic hypothesis of a single species of A. pallipes settling the Italian continental waters, is affected by complex evolutionary events. To solve the paradox, we hypothesized an evolutive scenario in which the separation of ancient mtDNA lineages likely occurred before the latest glacial periods. However, the speciation process remained incomplete due to secondary intensive postglacial contacts that forced the mingling of the genomes, and confounds the phylogeographic signature still detectable within mtDNA. Postglacial dispersion and the following demographic events, such as founder effects, drift and bottlenecks, abruptly depleted the local mtDNA variation, and shaped the current genetic population structure of white-clawed crayfish.     

Population structure of species: Eco-geographic units and genetic differentiation between populations

A two-step approach, based on a combined use of environmental, geographic, and genetic data, is suggested for studying population structures of species. First, populations are grouped into eco-geographic units (EGUs) according to the environmental gradients in the studied part of the species range, the types of life strategies, and other non-genetic characteristics that are presumably associated with adaptation and interpopulation gene flows. Second, the selected EGUs are tested for their congruence with genetic data by comparing the genetic differentiation between populations within EGUs to that between populations of different EGUs. Some of the issues discussed are as follows: the relationship of the EGU concept with the concepts of biogeocenosis and evolutionarily significant units (ESUs); designing EGUs in practice; the level of EGUs in a hierarchical population structure; and the weights of genetic and phenotypic markers in estimating population differentiation. The population structure of a salmonid fish, the Sakhalin taimen, in terms of eco-geographic units is considered as an example.     

Population genetic structure of the tropical tree species Aegiphila sellowiana (Lamiaceae)

The Tibagi River, located in southern Brazil, is associated with a significant degree of environmental heterogeneity, along its 550 km extension. There is concern about the integrity of this river's ecosystem, as human interference has been increasing. Aegiphila sellowiana (Lamiaceae) is an important pioneer tree species, commonly found near rivers; the fruit is consumed by avifauna. We studied this species along three ecological gradients, comprising the upper, middle, and lower regions of the Tibagi River basin. The genetic structure of nine subpopulations of A. sellowiana distributed along these gradients was investigated using RAPDs. Moderate levels of gene diversity (ranging from 0.091 to 0.132) were identified, inferred by a traditional approach and a Bayesian model-based method. The F-statistic, G(ST) parameters and molecular variance analysis showed high genetic differentiation among the three regions (39.5 to 50.26%). Analysis of molecular variance revealed high levels of genetic variation between populations (50.26%), while lower values of genetic variation (ranging from 9.56 to 16.35%) were seen between subpopulations within the upper, middle, and lower regions of the Tibagi River basin. The validity of these results was confirmed by principal coordinate analysis. Linear regression analysis showed significant correlations (r = 0.621, P = 0.0001) between the genetic and geographical distances. The differences observed in genetic variation between regions are probably due to habitat fragmentation; for conservation purposes, we recommend that at least one subpopulation from each region of the Tibagi River should be maintained.