Eco-geographically divergent diploids, Caucasian clover (Trifolium ambiguum) and western clover (T. occidentale), retain most requirements for hybridization

Background and Aims

DNA sequence similarities and hybridization patterns in Trifolium (clovers) section Trifoliastrum suggest that rapid radiation from a common ancestral source led to this complex of diverse species distributed across Europe, western Asia and North Africa. Two of the most geographically and ecologically divergent of these species are the rhizomatous T. ambiguum from high altitudes in eastern Europe and western Asia and the stoloniferous T. occidentale from sea level in western Europe. Attempts were made to hybridize these species to ascertain whether, despite this separation, gene flow could be achieved, indicating the retention of the genetic factors necessary for hybridization.

Methods

Three F₁ hybrids formed after embryo rescue were described, characterized by conventional and molecular cytogenetics, subjected to fertility tests and progeny generations were developed.

Results and Conclusions

Partially fertile hybrids between Trifolium ambiguum and T. occidentale were obtained for the first time. The F₁ hybrids produced seeds after open-pollination, and also produced triploid progeny in backcrosses to T. occidentale from the functioning of unreduced gametes in the hybrids. These plants were fertile and produced progeny with T. occidentale and with T. repens. Meiotic chromosome pairing in the F₁ showed six to eight bivalents per pollen mother cell, indicating pairing between the parental genomes. A chromosome-doubled form of one hybrid, produced using colchicine, showed some multivalents, indicative of interspecific chromosome pairing. The hybrid plants were robust and combined phenotypic characteristics of both species, having stolons, thick roots and a few rhizomes. Results show that despite separation by the entire breadth of Europe, the speciation process is incomplete, and these taxa have partially retained most of the genetic compatibilities needed for hybridization (possibly except for endosperm development, which was not tested). The fertile progeny populations could lead to new clover breeding strategies based on new hybrid forms.     

Genetic diversity and population structure inferred from the partially duplicated genome of domesticated carp,Cyprinus carpio L.

Genetic relationships among eight populations of domesticated carp (Cyprinus carpio L.), a species with a partially duplicated genome, were studied using 12 microsatellites and 505 AFLP bands. The populations included three aquacultured carp strains and five ornamental carp (koi) variants. Grass carp (Ctenopharyngodon idella) was used as an outgroup. AFLP-based gene diversity varied from 5% (grass carp) to 32% (koi) and reflected the reasonably well understood histories and breeding practices of the populations. A large fraction of the molecular variance was due to differences between aquacultured and ornamental carps. Further analyses based on microsatellite data, including cluster analysis and neighbor-joining trees, supported the genetic distinctiveness of aquacultured and ornamental carps, despite the recent divergence of the two groups. In contrast to what was observed for AFLP-based diversity, the frequency of heterozygotes based on microsatellites was comparable among all populations. This discrepancy can potentially be explained by duplication of some loci in Cyprinus carpio L., and a model that shows how duplication can increase heterozygosity estimates for microsatellites but not for AFLP loci is discussed. Our analyses in carp can help in understanding the consequences of genotyping duplicated loci and in interpreting discrepancies between dominant and co-dominant markers in species with recent genome duplication.     

Genetic diversity and population structure in contemporary house sparrow populations along an urbanization gradient

House sparrow (Passer domesticus) populations have suffered major declines in urban as well as rural areas, while remaining relatively stable in suburban ones. Yet, to date no exhaustive attempt has been made to examine how, and to what extent, spatial variation in population demography is reflected in genetic population structuring along contemporary urbanization gradients. Here we use putatively neutral microsatellite loci to study if and how genetic variation can be partitioned in a hierarchical way among different urbanization classes. Principal coordinate analyses did not support the hypothesis that urban/suburban and rural populations comprise two distinct genetic clusters. Comparison of F(ST) values at different hierarchical scales revealed drift as an important force of population differentiation. Redundancy analyses revealed that genetic structure was strongly affected by both spatial variation and level of urbanization. The results shown here can be used as baseline information for future genetic monitoring programmes and provide additional insights into contemporary house sparrow dynamics along urbanization gradients.     

Genetic diversity and population structure in the tomato-like nightshades Solanum lycopersicoides and S. sitiens

Background and Aims

Two closely related, wild tomato-like nightshade species, Solanum lycopersicoides and Solanum sitiens, inhabit a small area within the Atacama Desert region of Peru and Chile. Each species possesses unique traits, including abiotic and biotic stress tolerances, and can be hybridized with cultivated tomato. Conservation and utilization of these tomato relatives would benefit from an understanding of genetic diversity and relationships within and between populations.

Methods

Levels of genetic diversity and population genetic structure were investigated by genotyping representative accessions of each species with a set of simple sequence repeat (SSR) and allozyme markers.

Key Results

As expected for self-incompatible species, populations of S. lycopersicoides and S. sitiens were relatively diverse, but contained less diversity than the wild tomato Solanum chilense, a related allogamous species native to this region. Populations of S. lycopersicoides were slightly more diverse than populations of S. sitiens according to SSRs, but the opposite trend was found with allozymes. A higher coefficient of inbreeding was noted in S. sitiens. A pattern of isolation by distance was evident in both species, consistent with the highly fragmented nature of the populations in situ. The populations of each taxon showed strong geographical structure, with evidence for three major groups, corresponding to the northern, central and southern elements of their respective distributions.

Conclusions

This information should be useful for optimizing regeneration strategies, for sampling of the populations for genes of interest, and for guiding future in situ conservation efforts.     

Spatial genetic structure of Aquilegia taxa endemic to the island of Sardinia

Background and Aims

The Mediterranean Basin is one of the most important regions for the Earth''s plant biodiversity; however, the scarcity of studies on fine scale patterns of genetic variation in this region is striking. Here, an assessment is made of the spatial genetic structure of all known locations of the three Sardinian endemic species of Aquilegia in order to determine the relative roles of gene flow and genetic drift as underlying evolutionary forces canalizing the divergence of Sardinian Aquilegia taxa, and to see if the spatial genetic structure found fits the current taxonomic differentiation of these taxa.

Methods

DNA from 89 individuals from all known locations of Aquilegia across Sardinia was analysed by means of amplified fragment length polymorphism (AFLP) markers. Both principal co-ordinates analysis (PCoA) and Bayesian clustering analyses were used to determine the spatial genetic structure irrespective of any taxonomic affiliation. Historical effects of gene flow and genetic drift were assessed by checking for the existence of isolation-by-distance patterns.

Key Results

STRUCTURE and PCoA analyses revealed a pattern of genetic variation geographically structured into four spatial genetic groups. No migration–drift equilibrium was detected for Aquilegia in Sardinia, when analysed either as a whole or in individual groups. The scenario approached a Case III pattern sensu Hutchinson and Templeton, which is associated with extreme isolation conditions where genetic drift has historically played a dominant role over gene flow.

Conclusions

The pattern of genetic variation of Sardinian taxa of Aquilegia indicates that genetic drift has been historically more influential than gene flow on population structure of Sardinian species of Aquilegia. Limited seed dispersal and divergent selection imposed by habitat conditions have been probably the main causes reinforcing post-Pleistocene geographical isolation of Aquilegia populations. The spatial genetic structure found here is not fully compatible with current taxonomic affiliations of Sardinian Aquilegia taxa. This is probably a consequence of the uncoupling between morphological and genetic patterns of differentiation frequently found in recently radiated taxa.     

Variation and genetic structure of Melipona quadrifasciata Lepeletier (Hymenoptera, Apidae) populations based on ISSR pattern

For a study of diversity and genetic structuring in Melipona quadrifasciata, 61 colonies were collected in eight locations in the state of Minas Gerais, Brazil. By means of PCR analysis, 119 ISSR bands were obtained, 80 (68%) being polymorphic. H_e and H _B were 0.20 and 0.16, respectively. Two large groups were obtained by the UPGMA method, one formed by individuals from Januária, Urucuia, Rio Vermelho and Caeté and the other by individuals from São João Del Rei, Barbacena, Ressaquinha and Cristiano Otoni. The Φst and θ^B values were 0.65 and 0.58, respectively, thereby indicating high population structuring. UPGMA grouping did not reveal genetic structuring of M. quadrifasciata in function of the tergite stripe pattern. The significant correlation between dissimilarity values and geographic distances (r = 0.3998; p < 0.05) implies possible geographic isolation. The genetic differentiation in population grouping was probably the result of an interruption in gene flow, brought about by geographic barriers between mutually close geographical locations. Our results also demonstrate the potential of ISSR markers in the study of Melipona quadrifasciata population structuring, possibly applicable to the studies of other bee species.     

Distinct population structure for co-occurring Anopheles goeldii and Anopheles triannulatus in Amazonian Brazil

To evaluate whether environmental heterogeneity contributes to the genetic heterogeneity in Anopheles triannulatus, larval habitat characteristics across the Brazilian states of Roraima and Pará and genetic sequences were examined. A comparison with Anopheles goeldii was utilised to determine whether high genetic diversity was unique to An. triannulatus. Student t test and analysis of variance found no differences in habitat characteristics between the species. Analysis of population structure of An. triannulatus and An. goeldii revealed distinct demographic histories in a largely overlapping geographic range. Cytochrome oxidase I sequence parsimony networks found geographic clustering for both species; however nuclear marker networks depicted An. triannulatus with a more complex history of fragmentation, secondary contact and recent divergence. Evidence of Pleistocene expansions suggests both species are more likely to be genetically structured by geographic and ecological barriers than demography. We hypothesise that niche partitioning is a driving force for diversity, particularly in An. triannulatus.     

Interference effects in white clover genotypes grown as pure stands and binary mixtures with different grass species and varieties

Six white clover genotypes and eight grass varieties belonging to four different species were grown both in monoculture and as grass-legume binary mixtures in dense swards for two years under a mowing regime and a management including N fertilization. Dry matter yield and yield-related traits were recorded to investigate some aspects of inter-specific interference in white clover-based mixtures and to define a methodology for selecting genotypes of this clover suited to conditions of association. Clover was at a competitive disadvantage in most mixtures. Differences among grasses for aggressiveness were related more to variety vigour than to species. Clover compatibility proved specific only in relation to grass vigour. Variation among clovers for tolerance to competitive stress involved significant cross-over interactions passing from monoculture to severe stress conditions for clover yield and other traits, and was related positively to stolon density and negatively to yield and leaf gigantism traits recorded in monoculture. Clover selection for high levels of competitive stress seems possible either by genotype assessment in stress conditions or by a combination of high yield and stolon density assessed in monoculture.     

Effect of Meloidogyne incognita on Reproduction of Pratylenchus penetrans in Red Clover and Alfalfa

Roots of seedlings of red clover and alfalfa growing on 10⁻¹ Hoagland and Arnon solution agar were inoculated with various combinations of Meloidogyne incognita and Pratylenchus penetrans. Egg-laying by P. penetrans decreased as the number of nematodes, the ratio of entrant M. incognita to entrant P. penetrans, and the priority of invasion of roots by M. incognita increased. Embryogeny and hatching of eggs of P. penetrans, and development of larvae of M. incognita, were not affected. In red clover, the greatest red uction occurred when there were 65 entrant nematodes, the ratio of M. incognita:P. penetrans was 4:1 and M. incognita was inoculated four days prior to P. penetrans. In alfalfa, the less-favorable host for both nematodes, the greatest reduction occurred when there were 45 entrant nematodes, the ratio of M. incognita:P. penetrans was 2:1, and M. incognita was inoculated 4 days prior to P. penetrans.     

Transgenic white clover. Studies with the auxin-responsive promoter,GH3, in root gravitropism and lateral root development

We report an improved method for white clover (Trifolium repens) transformation usingAgrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. Thebar gene and phosphinothricin selection was shown to be a more effective selection system thannptII (kanamycin selection) oraadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.     

Effects of geographic distance,sea barriers and habitat on the genetic structure and diversity of all-hybrid water frog populations

The history of population size and migration patterns leaves its mark in the genetics of populations. We investigate the genetic structure of the edible frog, Pelophylax esculentus in the Danish archipelago and adjacent countries. This frog is of particular interest because it is a hybrid that, in this area, forms all-hybrid populations of diploid (LR) and triploid (LLR and LRR) genomotypes with no (or very few) adults of the parental species (LL and RR). This study is the first to cover the entire geographic range of Danish, Swedish and German all-hybrid populations, documenting their extent and providing a broad picture of their diversity of neutral genetic markers and genomotype proportions. With 18 microsatellite markers, we found that genetic diversity declines northwards in agreement with the glacial refuge and central-marginal hypotheses; however, populations on small and medium-sized islands are no less diverse than those on large islands and continental peninsulas. Isolation by distance exists across the archipelago with limited influence of fragmentation by brackish seawater. The extremely low genetic diversity in all-hybrid populations, compared with adjacent populations, may be responsible for the maintenance of their special breeding system. We also show large variation among ponds in proportions of LLR, LR and LRR genomotypes, but little geographic pattern in their distribution. Instead, we found relationships between the genomotype proportions and some of 15 habitat parameters monitored. Body size differences among LLR, LR and LRR further suggest ecological differences.     

Temporal variation in genetic diversity and effective population size of Mediterranean and subalpine Arabidopsis thaliana populations

Currently, there exists a limited knowledge on the extent of temporal variation in population genetic parameters of natural populations. Here, we study the extent of temporal variation in population genetics by genotyping 151 genome-wide SNP markers polymorphic in 466 individuals collected from nine populations of the annual plant Arabidopsis thaliana during 4 years. Populations are located along an altitudinal climatic gradient from Mediterranean to subalpine environments in NE Spain, which has been shown to influence key demographic attributes and life cycle adaptations. Genetically, A. thaliana populations were more variable across space than over time. Common multilocus genotypes were detected several years in the same population, whereas low-frequency multilocus genotypes appeared only 1 year. High-elevation populations were genetically poorer and more variable over time than low-elevation populations, which might be caused by a higher overall demographic instability at higher altitudes. Estimated effective population sizes were low but also showed a significant decreasing trend with increasing altitude, suggesting a deeper impact of genetic drift at high-elevation populations. In comparison with single-year samplings, repeated genotyping over time captured substantially higher amount of genetic variation contained in A. thaliana populations. Furthermore, repeated genotyping of populations provided novel information on the genetic properties of A. thaliana populations and allowed hypothesizing on their underlying mechanisms. Therefore, including temporal genotyping programmes into traditional population genetic studies can significantly increase our understanding of the dynamics of natural populations.     

Using genetic diversity information to establish core collections of Stylosanthes capitata and Stylosanthes macrocephala

Stylosanthes species are important forage legumes in tropical and subtropical areas. S. macrocephala and S. capitata germplasm collections that consist of 134 and 192 accessions, respectively, are maintained at the Brazilian Agricultural Research Corporation Cerrados (Embrapa-Cerrados). Polymorphic microsatellite markers were used to assess genetic diversity and population structure with the aim to assemble a core collection. The mean values of H_O and H_E for S. macrocephala were 0.08 and 0.36, respectively, whereas the means for S. capitata were 0.48 and 0.50, respectively. Roger’s genetic distance varied from 0 to 0.83 for S. macrocephala and from 0 to 0.85 for S. capitata. Analysis with STRUCTURE software distinguished five groups among the S. macrocephala accessions and four groups among those of S. capitata. Nei’s genetic diversity was 27% in S. macrocephala and 11% in S. capitata. Core collections were assembled for both species. For S. macrocephala, all of the allelic diversity was represented by 23 accessions, whereas only 13 accessions were necessary to represent all allelic diversity for S. capitata. The data presented herein evidence the population structure present in the Embrapa-Cerrados germplasm collections of S. macrocephala and S. capitata, which may be useful for breeding programs and germplasm conservation.     

An assessment of the AFLP method for investigating population structure in the red alga Chondrus crispus Stackhouse (Gigartinales, Florideophyceae)

The appropriateness of the Amplified Fragment Length Polymorphism (AFLP) technique for investigating Chondrus crispus Stackhouse populations in the Maritime Provinces of Canada was assessed. The AFLP procedure was first subjected to reproducibility testing and three shortcomings were noted: 1) failure to reproduce band intensity between replicate runs for the same individual and primer pair; 2) failure of some bands to replicate; 3) lack of reproducibility for complete replicate runs for some individuals and primer pairs. In the last-mentioned case, the lack of reproducibility resulted in characteristic electropherograms indicative of weak reactions. These weak runs can be attributed to poor restriction digest/ligation reactions and/or substandard PCR, these failures ultimately resulting from low and inconsistent DNA quality. We recommend that reproducibility testing should be completed routinely in studies using the AFLP technique. In the current work, only fragments and individuals that gave reproducible results were used in subsequent analyses. The AFLP method resulted in highly variable markers within and between the populations of C. crispus included in this investigation, which prevented successful resolution of population structure. This situation could result from a lack of suitability for AFLP markers in population genetic studies, and/or too extensive genetic variation for C. crispus populations to be discerned by the AFLP technique. These two possible explanations are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analysis of genetic population structure in Acacia caven (Leguminosae,Mimosoideae), comparing one exploratory and two Bayesian-model-based methods

Bayesian clustering as implemented in STRUCTURE or GENELAND software is widely used to form genetic groups of populations or individuals. On the other hand, in order to satisfy the need for less computer-intensive approaches, multivariate analyses are specifically devoted to extracting information from large datasets. In this paper, we report the use of a dataset of AFLP markers belonging to 15 sampling sites of Acacia caven for studying the genetic structure and comparing the consistency of three methods: STRUCTURE, GENELAND and DAPC. Of these methods, DAPC was the fastest one and showed accuracy in inferring the K number of populations (K = 12 using the find.clusters option and K = 15 with a priori information of populations). GENELAND in turn, provides information on the area of membership probabilities for individuals or populations in the space, when coordinates are specified (K = 12). STRUCTURE also inferred the number of K populations and the membership probabilities of individuals based on ancestry, presenting the result K = 11 without prior information of populations and K = 15 using the LOCPRIOR option. Finally, in this work all three methods showed high consistency in estimating the population structure, inferring similar numbers of populations and the membership probabilities of individuals to each group, with a high correlation between each other.     

Growth regulators differentially affect photosensitivity induced by low temperature and osmotic stresses in germinating white clover seeds

Negative photoblastism induced in white clover seeds at 5°C or by lowered water potential (–0.3 MPa, polyethylene glycol) was affected by ethrel, gibberellin A₃, benzylaminopurine and kinetin treatments. The effects were different for water and temperature stressed seeds. The observed synergistic and additive effects of light and growth regulators confirm the earlier suggestion that there are two different mechanisms involved in the light inhibition of white clover seed germination induced by various adverse environmental conditions.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellin A₃ - PEG polyethylene glycol     

Short-term genetic changes: evaluating effective population size estimates in a comprehensively described brown trout (Salmo trutta) population

The effective population size (N(e)) is notoriously difficult to accurately estimate in wild populations as it is influenced by a number of parameters that are difficult to delineate in natural systems. The different methods that are used to estimate N(e) are affected variously by different processes at the population level, such as the life-history characteristics of the organism, gene flow, and population substructure, as well as by the frequency patterns of genetic markers used and the sampling design. Here, we compare N(e) estimates obtained by different genetic methods and from demographic data and elucidate how the estimates are affected by various factors in an exhaustively sampled and comprehensively described natural brown trout (Salmo trutta) system. In general, the methods yielded rather congruent estimates, and we ascribe that to the adequate genotyping and exhaustive sampling. Effects of violating the assumptions of the different methods were nevertheless apparent. In accordance with theoretical studies, skewed allele frequencies would underestimate temporal allele frequency changes and thereby upwardly bias N(e) if not accounted for. Overlapping generations and iteroparity would also upwardly bias N(e) when applied to temporal samples taken over short time spans. Gene flow from a genetically not very dissimilar source population decreases temporal allele frequency changes and thereby acts to increase estimates of N(e). Our study reiterates the importance of adequate sampling, quantification of life-history parameters and gene flow, and incorporating these data into the N(e) estimation.     

Spatial and temporal population genetic variation and structure of Nothotsuga longibracteata (Pinaceae), a relic conifer species endemic to subtropical China

Nothotsuga longibracteata, a relic and endangered conifer species endemic to subtropical China, was studied for examining the spatial-temporal population genetic variation and structure to understand the historical biogeographical processes underlying the present geographical distribution. Ten populations were sampled over the entire natural range of the species for spatial analysis, while three key populations with large population sizes and varied age structure were selected for temporal analyses using both nuclear microsatellites (nSSR) and chloroplast microsatellites (cpSSR). A recent bottleneck was detected in the natural populations of N. longibracteata. The spatial genetic analysis showed significant population genetic differentiation across its total geographical range. Notwithstanding, the temporal genetic analysis revealed that the level of genetic diversity between different age class subpopulations remained constant over time. Eleven refugia of the Last Glacial Maximum were identified, which deserve particular attention for conservation management.     

Maintaining genetic diversity and population panmixia through dispersal and not gene flow in a holocyclic heteroecious aphid species

Heteroecious holocyclic aphids exhibit both sexual and asexual reproduction and alternate among primary and secondary hosts. Most of these aphids can feed on several related hosts, and invasions to new habitats may limit the number of suitable hosts. For example, the aphid specialist Aphis glycines survives only on the primary host buckthorn (Rhamnus spp.) and the secondary host soybean (Glycine max) in North America where it is invasive. Owing to this specialization and sparse primary host distribution, host colonization events could be localized and involve founder effects, impacting genetic diversity, population structure and adaptation. We characterized changes in the genetic diversity and structure across time among A. glycines populations. Populations were sampled from secondary hosts twice in the same geographical location: once after secondary colonization (early season), and again immediately before primary host colonization (late season). We tested for evidence of founder effects and genetic isolation in early season populations, and whether or not late-season dispersal restored genetic diversity and reduced fragmentation. A total of 24 single-nucleotide polymorphisms and 6 microsatellites were used for population genetic statistics. We found significantly lower levels of genotypic diversity and more genetic isolation among early season collections, indicating secondary host colonization occurred locally and involved founder effects. Pairwise F(ST) decreased from 0.046 to 0.017 in early and late collections, respectively, and while genetic relatedness significantly decreased with geographical distance in early season collections, no spatial structure was observed in late-season collections. Thus, late-season dispersal counteracts the secondary host colonization through homogenization and increases genetic diversity before primary host colonization.