Obligate Outbreeding Research Articles

Genomic Prediction of Complex Traits in Forage Plants Species: Perennial Grasses Case.

The majority of forage grass species are obligate outbreeders. Their breeding classically consists of an initial selection on spaced plants for highly heritable traits such as disease resistances and heading date, followed by familial selection on swards for forage yield and quality traits. The high level of diversity and heterozygosity, and associated decay of linkage disequilibrium (LD) over very short genomic distances, has hampered the implementation of genomic selection (GS) in these species. However, next generation sequencing technologies in combination with the development of genomic resources have recently facilitated implementation of GS in forage grass species such as perennial ryegrass (Lolium perenne L.), switchgrass (Panicum virgatum L.), and timothy (Phleum pratense L.). Experimental work and simulations have shown that GS can increase significantly the genetic gain per unit of time for traits with different levels of heritability. The main reasons are (1) the possibility to select single plants based on their genomic estimated breeding values (GEBV) for traits measured at sward level, (2) a reduction in the duration of selection cycles, and less importantly (3) an increase in the selection intensity associated with an increase in the genetic variance used for selection. Nevertheless, several factors should be taken into account for the successful implementation of GS in forage grasses. For example, it has been shown that the level of relatedness between the training and the selection population is particularly critical when working with highly structured meta-populations consisting of several genetic groups. A sufficient number of markers should be used to estimate properly the kinship between individuals and to reflect the variability of major QTLs. It is also important that the prediction models are trained for relevant environments when dealing with traits with high genotype×environment interaction (G×E). Finally, in these outbreeding species, measures to reduce inbreeding should be used to counterbalance the high selection intensity that can be achieved in GS.

Odour-based social recognition in Damaraland mole-rats, Fukomys damarensis

The ability to discriminate between conspecifics functions in mate choice, kin-selected cooperation and territory defence. In mammals, olfaction plays a key role in such social interactions. Olfactory cues may be particularly important for subterranean mammals, for which visual and acoustic cues are less effective. Damaraland mole-rats live in groups comprising a breeding pair and their nonbreeding offspring. They are xenophobic, obligate outbreeders and independent dispersal represents the usual route to reproduction for both sexes. As yet, little is known about how dispersing individuals locate mates. Using a series of behavioural experiments, we reveal that mole-rats can discriminate between unfamiliar breeding groups and solitary, nonbreeders of the opposite sex by using odour cues. Our experiments showed that subjects spent more time investigating sand taken from other mole-rat groups than control sand, indicating an ability to recognize substrate-borne conspecific odours. Mole-rats also spent more time digging and sweeping in sand taken from the tunnels of unfamiliar, solitary animals of the opposite sex than sand taken from unfamiliar breeding groups and removed a higher volume of this sand during the experiments. Together, these results suggest an olfactory preference in both sexes for solitary, opposite-sex animals over breeding groups. Our results are supported by observations from the field that immigration into breeding groups is rare, with dispersing females typically establishing new groups, where they are subsequently joined by unfamiliar males. This study supports olfaction as a potential recognition cue which may facilitate adaptive dispersal. • Damaraland mole-rats recognize conspecifics using substrate-borne odour cues. • Both sexes dig the most in sand from the tunnels of unfamiliar, opposite-sex mole-rats. • Digging effort is reduced in sand taken from the tunnels of other breeding groups. • A primary role for olfaction in social recognition is supported in this species.

Genetic diversity in British populations of Taxus baccata L.: Is the seedbank collection representative of the genetic variation in the wild?

Seed banking is an invaluable tool in plant conservation, both as an archive and a source of genetic variation. Despite the increasing focus on the validation of sampling strategies, few studies have empirically related diversity in seed bank collections with variation in wild-provenance populations. By using a set of nuclear microsatellites, we investigated genetic diversity in British populations of Taxus baccata (European yew tree). We used our findings as a baseline for the quantification of genetic diversity captured in the germplasm collections maintained by the Millennium Seed Bank (Royal Botanic Gardens, Kew), in terms of allelic richness and allelic capture. We observed that genetic differentiation in wild populations of yew is compatible with highly effective gene flow, with no geographic patterns. Heterozygosity is lower than expected for a dioecious obligate outbreeder. Seed collections are representative of wild populations in terms of allelic capture, including rare and locally common variants, indicating that the current sampling protocol implemented by the UK National Tree Seed Project is appropriate. Specific sampling strategies might be improved by including more populations at the edge of the range and in remote localities, even at the expense of contiguous populations. Our methods may be applied to evaluate allelic capture in other germplasm collections for which a baseline exists, i.e. in which genetic diversity in the wild-provenance populations is known. Our recommendations related to sampling strategies can be extended to other tree species with continuous distributions and effective gene flow.

Environmental filtering predicts plant-community trait distribution and diversity: Kettle holes as models of meta-community systems.

Meta‐communities of habitat islands may be essential to maintain biodiversity in anthropogenic landscapes allowing rescue effects in local habitat patches. To understand the species‐assembly mechanisms and dynamics of such ecosystems, it is important to test how local plant‐community diversity and composition is affected by spatial isolation and hence by dispersal limitation and local environmental conditions acting as filters for local species sorting.We used a system of 46 small wetlands (kettle holes)—natural small‐scale freshwater habitats rarely considered in nature conservation policies—embedded in an intensively managed agricultural matrix in northern Germany. We compared two types of kettle holes with distinct topographies (flat‐sloped, ephemeral, frequently plowed kettle holes vs. steep‐sloped, more permanent ones) and determined 254 vascular plant species within these ecosystems, as well as plant functional traits and nearest neighbor distances to other kettle holes.Differences in alpha and beta diversity between steep permanent compared with ephemeral flat kettle holes were mainly explained by species sorting and niche processes and mass effect processes in ephemeral flat kettle holes. The plant‐community composition as well as the community trait distribution in terms of life span, breeding system, dispersal ability, and longevity of seed banks significantly differed between the two habitat types. Flat ephemeral kettle holes held a higher percentage of non‐perennial plants with a more persistent seed bank, less obligate outbreeders and more species with seed dispersal abilities via animal vectors compared with steep‐sloped, more permanent kettle holes that had a higher percentage of wind‐dispersed species. In the flat kettle holes, plant‐species richness was negatively correlated with the degree of isolation, whereas no such pattern was found for the permanent kettle holes.Synthesis: Environment acts as filter shaping plant diversity (alpha and beta) and plant‐community trait distribution between steep permanent compared with ephemeral flat kettle holes supporting species sorting and niche mechanisms as expected, but we identified a mass effect in ephemeral kettle holes only. Flat ephemeral kettle holes can be regarded as meta‐ecosystems that strongly depend on seed dispersal and recruitment from a seed bank, whereas neighboring permanent kettle holes have a more stable local species diversity.

Paternally expressed imprinted genes associate with hybridization barriers in Capsella.

Hybrid seed lethality is a widespread type of reproductive barrier among angiosperm taxa1,2 that contributes to species divergence by preventing gene flow between natural populations3,4. Besides its ecological importance, it is an important obstacle to plant breeding strategies 5 . Hybrid seed lethality is mostly due to a failure of the nourishing endosperm tissue, resulting in embryo arrest3,6,7. The cause of this failure is a parental dosage imbalance in the endosperm that can be a consequence of either differences in parental ploidy levels or differences in the 'effective ploidy', also known as the endosperm balance number (EBN)8,9. Hybrid seed defects exhibit a parent-of-origin pattern3,6,7, suggesting that differences in number or expression strength of parent-of-origin-specific imprinted genes underpin, as the primary or the secondary cause, the molecular basis of the EBN7,10. Here, we have tested this concept in the genus Capsella and show that the effective ploidy of three Capsella species correlates with the number and expression level of paternally expressed genes (PEGs). Importantly, the number of PEGs and the effective ploidy decrease with the selfing history of a species: the obligate outbreeder Capsella grandiflora had the highest effective ploidy, followed by the recent selfer Capsella rubella and the ancient selfer Capsella orientalis. PEGs were associated with the presence of transposable elements and their silencing mark, DNA methylation in CHH context (where H denotes any base except C). This suggests that transposable elements have driven the imprintome divergence between Capsella species. Together, we propose that variation in transposable element insertions, the resulting differences in PEG number and divergence in their expression level form one component of the effective ploidy variation between species of different breeding system histories, and, as a consequence, allow the establishment of endosperm-based hybridization barriers.

Population structure of the ash dieback pathogen, Hymenoscyphus fraxineus, in relation to its mode of arrival in the UK.

The ash dieback fungus, Hymenoscyphus fraxineus, a destructive, alien pathogen of common ash (Fraxinus excelsior), has spread across Europe over the past 25 years and was first observed in the UK in 2012. To investigate the relationship of the pathogen's population structure to its mode of arrival, isolates were obtained from locations in England and Wales, either where established natural populations of ash had been infected by wind‐dispersed ascospores or where the fungus had been introduced on imported planting stock. Population structure was determined by tests for vegetative compatibility (VC), mating type and single‐nucleotide polymorphisms (SNPs). VC heterogeneity was high at all locations, with 96% of isolate pairings being incompatible. Frequencies of the MAT1‐1‐1 and MAT1‐2‐1 idiomorphs were approximately equal, consistent with H. fraxineus being an obligate outbreeder. Most SNP variation occurred within study location and there was little genetic differentiation between the two types of location in the UK, or between pathogen populations in the UK and continental Europe. There was modest differentiation between UK subpopulations, consistent with genetic variation between source populations in continental Europe. However, there was no evidence of strong founder effects, indicating that numerous individuals of H. fraxineus initiated infection at each location, regardless of the route of pathogen transmission. The ssRNA virus HfMV1 was present at moderate to high frequencies in all UK subpopulations. The results imply that management of an introduced plant pathogen requires action against its spread at the continental level involving coordinated efforts by European countries.

Reproductive Assurance through Autogamous Self-pollination across Diverse Sexual and Breeding Systems

Pollination becomes a constraint when conspecific plants and/or their pollinators become scarce. Many plant species have evolved autogamous self-pollination as a means of reproductive assurance (RA) under pollination-uncertain environments. So far RA has been studied and discussed largely with reference to self-compatible species producing bisexual flowers. RA seems to have evolved across all other sexual and breeding systems - monoecy, dioecy and self-incompatibility (SI). Both monoecy and dioecy produce bisexual flowers (andro/gyno-monoecy, andro/gynodioecy and polygamous conditions) which may provide RA. Similarly, most of the SI species are leaky and do set some seeds upon self-pollination. This phenomenon termed 'partial self-compatibility' is quite common and does provide RA in SI species. Although dioecy and SI have evolved as obligate outbreeding systems, they seem to have reached an evolutionary dead end because of the constraints for outcross pollination. In the light of habitat destruction leading to a reduction in the diversity and density of native pollinators, it is likely that many of the obligate outbreeders tend to shift to mixed mating system in the coming decades. Similarly, obligate mutualism in which each plant species is dependent on one animal species for pollination also seems to have reached a dead end and the trend is to abandon such obligate mutualism as a survival strategy. In the absence of such a change, obligate out breeders and those with highly specialized pollination system are likely to become endangered or even extinct.

Pollination Strategies of Some Perennial Weed Species

Weeds have evolved a number of adaptations to thrive even under adverse conditions. One of the adaptations is their ability to set seeds even under pollination uncertain environments through autogamous self-pollination. Autogamous pollination which provides reproductive assurance (RA) is critical in annual weeds as they get only one chance to set seeds in their life; if they miss this chance their survival is threatened. In perennial weeds, however, RA through autogamous self-pollination is not so critical as they get repeated chances to set seeds; lack of seed set in some years does not affect their survival. There is very little information on pollination strategies of perennial weeds particularly of Indian species. To test this prediction, studies were carried out on five Indian perennial weeds — Cassia auriculata, Ipomoea obscura, Oxalis corniculata, Plumbago zeylanica and Dodonaea viscosa. Seed set in the populations of Ipomoea, Oxalis and Plumbago was exclusively dependent on autogamous pollination as they did not attract any pollinators and there was no difference in the extent of seed set in bagged and open-pollinated flowers. Autogamy was absent in Cassia; it depended exclusively on pollination by Xylocopa sp. Dodonaea depended on wind for pollen transfer. Thus, in agreement with the expectation, autogamy is not critical in perennial weed species and their pollination strategies vary from complete autogamy to obligate outbreeding, similar to non-weedy species.

Floral biology of large-flowered Thelymitra species (Orchidaceae) and their hybrids in Western Australia

Historically, only a few large flowered species in the genus Thelymitra were identified as obligate out-breeders. We compared floral presentation, pollen-pistil interactions, pollination ecology, and interspecific hybridization in two populations of T. macrophylla where its flowering periods overlapped with T. antennifera (Tenterden) and T. crinita (Lesmurdie) respectively. Pollen-pistil interactions were studied using glasshouse collections of T. crinita and T. macrophylla at the Kings Park and Botanic Garden (Perth, WA). The number of flowers/inflorescence in T. macrophylla varied significantly between sites. Climatic conditions influenced flower opening and closing regimes differently in T. crinita vs. T. macrophylla. While all three Thelymitra spp. opened on warm, sunny mornings and closed by late afternoon, T. crinita at Lesmurdie was significantly more likely to open its perianth segments on cool days compared to the co-blooming, sympatric flowers of T. macrophylla. The floral lifespans of individual flowers of T. macrophylla and T. crinita were reduced significantly following application of Thelymitra pollen onto the stigmatic surface but were not reduced by pollinarium removal. Flowers of both species were self-compatible but neither species self-pollinated mechanically (autogamy). Fluorescence microscopy also showed that both species were inter-compatible. Natural rates of pollinarium removal by insects were low in all three species at both sites. Natural rates of pollen deposition on receptive stigmas were significantly higher in T. crinita vs. T. macrophylla but pollen deposition was less than 12% in both species. Observations and collections of pollinators were infrequent and pollinaria vectors were restricted to a few polylectic, female bees in the families Apidae, Colletidae, and Halictidae. We found a large number of hybrids between T. antennifera and T. macrophylla at Tenterden but few obvious hybrids between T. crinita and T. macrophylla at Lesmurdie. As expected, all hybrids showed characteristics intermediate between their two putative parent species, including pollen configuration in the T. crinita X T. macrophylla specimens. Due to malformations of the column, the majority of T. antennifera X T. macrophylla flowers appeared unable to attach their pollinia to their respective rostella.

Development and implementation of a multiplexed single nucleotide polymorphism genotyping tool for differentiation of ryegrass species and cultivars

Perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.) are important temperate forage grasses which are closely related, generating fertile interspecific hybrids. All groups are represented by multiple cultivars in the commercial pasture seeds market. Due to the close taxonomic relationship between the two species, differentiation based on morphophysiological criteria is not always readily achievable. In addition, an obligate outbreeding reproductive habit produces high levels of individual heterozygosity and intrapopulation diversity, which presents problems for discrimination between cultivars. Molecular genetic marker polymorphism provides an effective means of addressing these challenges. An iterative process of resequencing from loci distributed across the perennial ryegrass genome was used to identify single nucleotide polymorphism (SNP) markers, which were then validated and formatted in a highly multiplexed (384-plex) assay system. SNP genotyping was then performed across samples of 48–192 individuals from a total of 27 ryegrass cultivars (19 of perennial ryegrass, seven of Italian ryegrass and one hybrid cultivar). SNP markers from perennial ryegrass exhibited a high level of transfer to Italian ryegrass. Data analysis permitted quantification of intra- and inter-species diversity, as well as discrimination between cultivars within each species, including diploid and autotetraploid cultivars of perennial ryegrass. Lower levels of SNP-based diversity were detected in Italian ryegrass than in perennial ryegrass. A neighbour-joining tree based on genetic distance analysis located a hybrid cultivar to an intermediate position between the two species-specific cultivar groups. The resulting catalogue of ryegrass cultivars will provide support for the processes of cultivar accreditation and quality assurance.

Characterisation of thirteen polymorphic microsatellite markers for cowslip (Primula veris L.) developed using a 454 sequencing approach

The cowslip (Primula veris L., Primulaceae) is a distylous perennial plant with a self-incompatibility system making it an obligate outbreeder. It is found in threatened grassland habitats that are becoming increasingly frag- mented. Microsatellite discovery was performed using next generation sequence technologies. We present 13 novel microsatellite markers that have been optimised for P. veris. The loci show clear polymorphic banding patterns, exhib- iting between 2 and 12 alleles, with observed heterozygosity values ranging from 0.296 to 0.889. These markers will facilitate the assessment of genetic diversity and population structure of P. veris populations.

Candidate gene-based association genetics analysis of herbage quality traits in perennial ryegrass (Lolium perenne L.)

Due to the complex genetic architecture of perennial ryegrass, based on an obligate outbreeding reproductive habit, association-mapping approaches to genetic dissection offer the potential for effective identification of genetic marker–trait linkages. Associations with genes for agronomic characters, such as components of herbage nutritive quality, may then be utilised for accelerated cultivar improvement using advanced molecular breeding practices. The objective of the present study was to evaluate the presence of such associations for a broad range of candidate genes involved in pathways of cell wall biosynthesis and carbohydrate metabolism. An association-mapping panel composed from a broad range of non-domesticated and varietal sources was assembled and assessed for genome-wide sequence polymorphism. Removal of significant population structure obtained a diverse meta-population (220 genotypes) suitable for association studies. The meta-population was established with replication as a spaced-plant field trial. All plants were genotyped with a cohort of candidate gene-derived single nucleotide polymorphism (SNP) markers. Herbage samples were harvested at both vegetative and reproductive stages and were measured for a range of herbage quality traits using near infrared reflectance spectroscopy. Significant associations were identified for ~50% of the genes, accounting for small but significant components of phenotypic variance. The identities of genes with associated SNPs were largely consistent with detailed knowledge of ryegrass biology, and they are interpreted in terms of known biochemical and physiological processes. Magnitudes of effect of observed marker–trait gene association were small, indicating that future activities should focus on genome-wide association studies in order to identify the majority of causal mutations for complex traits such as forage quality.

The Pollination Biology of an Annual Endemic Herb, Physaria filiformis (Brassicaceae), in the Missouri Ozarks Following Controlled Burns1

Physaria filiformis, a threatened species of calcareous glade communities, blooms profusely within the Rocky Barrens Conservation Area (Missouri) under a program of controlled burns. Flowers visited by insect foragers in 2008 and 2009 had a mean > 35 and > 50 germinating grains on their stigmas respectively and a mean of 1.96–2.0 pollen tubes entering their ovaries (mean of 4 ovules/ovary). Results of controlled hand-pollinations conducted in situ in 2009 showed that this species had a negligible rate of self-pollination (mechanical autogamy) and showed classic Brassica-type, early-acting self-incompatibility. Each dissected flower had nectar glands and each seasonal population was visited by insects representing four insect orders; Coleoptera, Diptera, Lepidoptera and Hymenoptera. The most commonly collected floral foragers found to carry the pollen of the host flower were male and female native bees representing 38 species in the Andrenidae, Apidae, Colletidae, Halictidae, Megachilidae and the naturalized Apis mellifera. Only four of these species (11%) were collected in all three years, but these four accounted for slightly more than a third of all individual bees collected. None of the bees were known specialists on flowers of the Brassicaceae and only two species were habitat (glade) specialists. As P. filiformis is an obligate out-breeder but a generalist entomophile we suggest that future management plans include conservation and management of adjacent non-glade habitats to sustain the diverse range of pollinators.

Extremely high molecular diversity within the East Asian nematode Caenorhabditis sp. 5

Most relatives of the self-fertilizing hermaphroditic nematode model organism Caenorhabditis elegans reproduce via obligate outbreeding between males and females, which also represents the ancestral mode of reproduction within the genus. However, little is known about the scope of genetic diversity and differentiation within such gonochoristic species, especially those found outside of temperate Europe and North America. It is critical to understand the evolutionary processes operating in these species to provide a framework for deciphering the evolution of hermaphroditism and a baseline for the application of outcrossing Caenorhabditis to problems in evolutionary genetics. Here, we investigate for the first time molecular sequence variation for Caenorhabditis sp. 5, a species found commonly in eastern Asia. We identify enormous levels of standing genetic variation that approach the levels observed in the marine broadcast-spawning sea squirt, Ciona savignyi. Although we document significant isolation by distance, we demonstrate that the high polymorphism within C. sp. 5 is not because of strong differentiation among populations or to the presence of cryptic species. These findings illustrate that molecular population genetic approaches to studying obligately outbreeding species of Caenorhabditis will prove powerful in identifying and characterizing functionally and evolutionarily important features of the genome.

Fine-scale comparative genetic and physical mapping supports map-based cloning strategies for the self-incompatibility loci of perennial ryegrass (Lolium perenne L.)

Perennial ryegrass is an obligate outbreeding pasture grass of the Poaceae family, with a two-locus (S and Z) gametophytic self-incompatibility (SI) mechanism. This system has provided a major obstacle to targeted varietal development, and enhanced knowledge is expected to support more efficient breeding strategies. Comparative genetics and physical mapping approaches have been developed to permit molecular cloning of the SI genes. SI gene-linked genetic markers based on heterologous cDNA restriction fragment length polymorphisms (RFLPs) and homologous genomic DNA-derived simple sequence repeats (SSRs) were converted to single nucleotide polymorphism (SNP) format for efficient genotyping. Genetic mapping identified the location of SI loci and demonstrated macrosynteny between related grass species. S- and Z-linked bacterial artificial chromosome (BAC) clones were sequenced using massively parallel pyrosequencing technology to provide the first physical mapping data for Poaceae SI loci. The sequence assembly process suggested a lower prevalence of middle repetitive sequences in the Z locus region and hence precedence for positional cloning strategy. In silico mapping using data from rice, Brachypodium distachyon and Sorghum revealed high sequence conservation in the vicinity of the Z locus region between SI and self-compatible (SC) grass species. Physical mapping identified a total of nine genes encoded in the Z locus region. Expression profiling and nucleotide diversity assessment identified two Z-linked genes, LpTC116908 and LpDUF247, as plausible candidates for the male and female determinants of the S-Z SI system.

Expression of Distinct Self-Incompatibility Specificities inArabidopsis thaliana

The interplay of balancing selection within a species and rapid gene evolution between species can confound our ability to determine the functional equivalence of interspecific and intergeneric pairs of alleles underlying reproduction. In crucifer plants, mating specificity in the barrier to self-fertilization called self-incompatibility (SI) is controlled by allele-specific interactions between two highly polymorphic and co-evolving proteins, the S-locus receptor kinase (SRK) and its S-locus cysteine rich (SCR) ligand. These proteins have diversified both within and between species such that it is often difficult to determine from sequence information alone if they encode the same or different SI specificity. The self-fertile Arabidopsis thaliana was derived from an obligate outbreeding ancestor by loss of self-incompatibility, often in conjunction with inactivation of SRK or SCR. Nevertheless, some accessions of A. thaliana can express self-incompatibility upon transformation with an SRK-SCR gene pair isolated from its self-incompatible close relative A. lyrata. Here we show that several additional and highly diverged SRK/SCR genes from A. lyrata and another crucifer plant, Capsella grandiflora, confer self-incompatibility in A. thaliana, either as intact genes isolated from genomic libraries or after manipulation to generate chimeric fusions. We describe how the use of this newly developed chimeric protein strategy has allowed us to test the functional equivalence of SRK/SCR gene pairs from different taxa and to assay the functionality of endogenous A. thaliana SRK and SCR sequences.

Biological Flora of the British Isles: Primula vulgaris Huds. (P. acaulis (L.) Hill)

1. This account presents information on all aspects of the biology of Primula vulgaris Huds. (Primrose) that are relevant to understanding its ecological characteristics and behaviour. The main topics are presented within the framework of the Biological Flora of the British Isles: distribution, habitat, communities, responses to biotic factors, responses to environment, structure and physiology, phenology, floral and seed characteristics, herbivores and disease, history and conservation. 2. Primula vulgaris is a native perennial herb with a very wide distribution in the British Isles. In many lowland areas it is essentially a plant of woodlands and hedgerows, although in the west it can occur abundantly in grasslands and other communities such as heaths. In northern and western Britain and Ireland, it may be frequent in open and even exposed habitats. Its distribution is linked with soil moisture and atmospheric humidity. It is shade tolerant, but it flowers most profusely in canopy gaps. 3. Primula vulgaris is a rosette hemicryptophyte that reproduces mainly through seeds. Vegetative spread is restricted and only occurs within very short distances from the mother plant through the production of lateral rosettes. Although individual rosettes may die, plants are relatively long-lived (life expectancy of a newborn individual: 48.3 years). The mean age at first flowering was 20 months. 4. Pollination is mainly by bumblebees and other bees, but other long-tongued pollinators including syrphids, bee-flies and even butterflies may be locally important. P. vulgaris is an obligate outbreeder, with two genetically determined self-incompatible morphs ('pin' and 'thrum'). A third morph ('homostyle' or 'long homostyle'), with a stigma like that of pin but anthers like that of thrum, has been found in Somerset and North Dorset. In years when pollinators are scarce, homostyles have higher reproductive success than pins and thrums, suggesting that reproductive assurance could have had a profound effect on the evolution of homostyly in P. vulgaris. 5. Seeds are dispersed by ants and rodents. Dispersal is usually restricted to a few centimetres or decimetres from maternal plants, resulting in significant fine-scale spatial genetic structure and small neighbourhood sizes. Pollen flow, on the other hand, is more extensive, but still limited to a few metres from paternal plants. 6. Populations of P. vulgaris have not changed markedly during the last century in most parts of Britain and Ireland and it is not threatened with extinction nationally. However, there is evidence that Primroses were formerly more widespread and have decreased with the decline of coppicing and pollarding. In East Anglia, woodland populations have declined greatly in response to a series of hot, dry summers since 1970. Small, isolated populations of P. vulgaris often show reduced reproductive success, potentially affecting their long-term survival. Preservation of local habitat conditions and restoration of gene flow among populations are required to maintain viable populations of P. vulgaris in the long term.

Biological Flora of the British Isles: Primula veris L.

Summary This account presents information on all aspects of the biology of Primula veris that are relevant to understanding its ecological characteristics and behaviour. The main topics are presented within the standard framework of the Biological Flora of the British Isles: distribution, habitat, communities, responses to biotic factors, responses to environment, structure and physiology, phenology, floral and seed characters, herbivores and diseases, history and conservation. Primula veris is a distylous, perennial rosette hemicryptophyte, mostly found in well‐drained, herb‐rich meadows and grasslands, in scrub or woodland rides and edges, and on calcareous cliffs. It occurs on base‐rich loams or clays, on limestone, and sometimes in dunes. Native to the British Isles, it is characteristic of many mesotrophic and calcicolous lowland grassland communities, but it also extends into upland valleys in northern England and Scotland. In Ireland it is also native and most abundant in the central plain. Primula veris occurs throughout the temperate areas of Europe as far east as the Russian border. Primula veris is a shade‐intolerant species that shows reduced performance and flowering under shade or increased competition. The species is not able to penetrate anoxic soil layers and is therefore mostly absent from locations that are characterized by a high water table. It has a well‐developed drought tolerance. Primula veris is an obligate outbreeder which entirely depends on foraging insects for successful pollination. Although the species shows heterostyly, with two reciprocal morphs (pin and thrum), only relatively low levels of intermorph pollination have been reported. Seed dispersal is restricted to a few centimetres from maternal plants, whereas pollen flow is wider, but still limited to a few metres from parental plants. Both are factors that contribute to a significantly fine‐scale spatial genetic structure and small neighbourhood size. At several locations throughout the British Isles, P. veris occurs together with P. elatior and especially P. vulgaris. The hybrid P. veris × P. vulgaris = P. × polyantha occurs frequently in mixed populations, whereas the hybrid P. veris × P. elatior = P. × media has been rarely reported in Britain. Although P. veris is still a widespread grassland herb, it is less abundant in the British Isles and continental Europe than before. This decline can be attributed to changes in land‐use practices, such as the loss of traditional hay–meadow management, the loss of grazing and an increase of ploughing, in combination with ongoing destruction and nutrient enrichment of permanent grasslands. However, from 1980 onwards the species has increased substantially in many areas of the UK, largely because its seeds are included in wildflower seed‐mixtures that are sown on new or upgraded road and motorway verges, embankments and urban conservation areas.

Biological Flora of the British Isles: Primula elatior (L.) Hill

Summary This account presents information on all aspects of the biology of Primula elatior that are relevant to understanding its ecological characteristics and behaviour. The main topics are presented within the standard framework of the Biological Flora of the British Isles: distribution, habitat, communities, responses to biotic factors, responses to environment, structure and physiology, phenology, floral and seed characters, herbivores and disease, history, and conservation. Primula elatior is a native perennial herb, within a compact area in the British Isles, in ancient woods and copses on the chalky boulder clay in East Anglia, where it is abundant locally although scarce nationally. British material is ssp. elatior, which extends across much of Europe, becoming rarer towards the south. Primula elatior is a shade tolerant species, but only flowers in profusion in the increased light levels following tree felling in traditionally coppiced woods. It is confined to an area of the British Isles where the soils are seasonally waterlogged and ill‐aerated, and is very tolerant of the toxic levels of ferrous iron which accumulate in the surface horizons in the spring months. It has a poorly developed drought tolerance. An early flowering plant, P. elatior is an insect‐pollinated obligate outbreeder, with two self‐incompatible morphs (pin and thrum). The low colonizing ability of this ancient woodland species can be attributed to both seed dispersal and seedling recruitment limitation. There has been a steady reduction in the density of P. elatior in woodland sites during the past centuries. However, there has been little change in the 10‐km distribution in the British Isles. Studies of populations involve three groups of factors: genetic variation, environmental changes and population structure. Potentially these factors have a greater effect on small and isolated populations than on large and integrated ones. There is no evidence that the species suffers from a loss of genetic diversity in the smaller populations. Primula elatior seems to have a relatively high level of genetic diversity that is maintained despite a fragmented distribution. Within its restricted distribution area P. elatior replaces P. vulgaris completely. At the margins of their distributions the two species occur together in a number of woodland sites, providing the opportunity for hybridization. As they are highly interfertile, hybridization is restricted mainly by differences in their habitat preferences. The hybrid P. vulgaris × P. elatior = P. × digenea is fertile and gives rise to hybrid swarms which are found in all British sites where both parents occur together. Changes in climate, woodland management practices and intensities of deer grazing, especially by Fallow deer (Dama dama), are likely to have a major effect on populations of P. elatior. Since the species colonizes new or secondary woodlands very slowly, management should focus on the conservation of ancient semi‐natural woodlands.

Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.