Using genetic evaluation to guide conservation of remnant Juniperus communis (Cupressaceae) populations.

Many critically endangered plant species exist in small, genetically depauperate or inbred populations, making assisted gene flow interventions necessary for long-term population viability. However, before such interventions are implemented, conservation practitioners must consider the genetic and demographic status of extant populations, which are strongly affected by species' life-history traits. In northwestern Europe, Juniperus communis, a dioecious, wind-pollinated and bird-dispersed gymnosperm, has been declining for the past century and largely exists in small, isolated and senescent populations. To provide useful recommendations for a recovery plan involving translocation of plants, we investigated genetic diversity and structure of populations in Belgium using four microsatellite and five plastid single-nucleotide polymorphism (SNP) markers. We detected no clonality in the populations, suggesting predominantly sexual reproduction. Populations exhibited high genetic diversity (He  = 0.367-0.563) and low to moderate genetic differentiation (FST  ≤ 0.133), with no clear geographic structure. Highly positive inbreeding coefficients (FIS  = 0.221-0.507) were explained by null alleles, population substructuring and biparental inbreeding. No isolation by distance was observed among distant populations, but isolation at close geographic proximity was found. Patterns were consistent with high historical gene flow through pollen and seed dispersal at both short and long distances. We also tested four pre-germination treatments among populations to improve germination rates; however, germination rates remained low and only cold-stratification treatments induced germination in some populations. To bolster population regeneration, introductions of cuttings from several source populations are recommended, in combination with in situ management practices that improve seedling survival and with ex situ propagation.

Temporal dynamics of sexual dimorphism in a dioecious species.

BACKGROUND AND AIMS: Sexual dimorphism for floral traits is common in dioecious plant species. Beyond its significance for understanding how selection acts on plant traits through male vs. female reproductive function, sexual dimorphism has also been proposed as a possible risky characteristic for insect-pollinated plants, as it could drive pollinators to forage mostly on male plants. However, even though most flowering plant species spread their flowering across several weeks or months, the temporal variation of floral phenotypes and sexual dimorphism have rarely been investigated. METHODS: We performed a survey of male and female plants from the dioecious generalist-pollinated Silene dioica (Caryophyllaceae) in a common garden experiment, over two consecutive flowering seasons. Flower number and floral size were measured each week, as well as pollen quantity and viability in male plants. KEY RESULTS: Sexual dimorphism was found for all investigated floral traits, with males showing an overall higher investment in flower production and flower size. Males and females showed a similar temporal decline in flower size. The temporal dynamics of daily flower number differed between sexes, with males showing a peak in the middle of their flowering season, whereas flower production by females was quite stable over time. At the scale of the experimental population, both individual and floral sex ratios appeared to vary across the flowering season. Moreover, because the onset of flowering varied among plants, the magnitude of sexual dimorphism in floral size also fluctuated strongly through time. CONCLUSIONS: Capturing male/female differences with only one temporal measurement per population may not be informative. This opens stimulating questions about how pollinator behaviour and resulting pollination efficiency may vary across the flowering season.

Male flowers of Aconitum compensate for toxic pollen with increased floral signals and rewards for pollinators.

Many plants require animal pollinators for successful reproduction; these plants provide pollinator resources in pollen and nectar (rewards) and attract pollinators by specific cues (signals). In a seeming contradiction, some plants produce toxins such as alkaloids in their pollen and nectar, protecting their resources from ineffective pollinators. We investigated signals and rewards in the toxic, protandrous bee-pollinated plant Aconitum napellus, hypothesizing that male-phase flower reproductive success is pollinator-limited, which should favour higher levels of signals (odours) and rewards (nectar and pollen) compared with female-phase flowers. Furthermore, we expected insect visitors to forage only for nectar, due to the toxicity of pollen. We demonstrated that male-phase flowers emitted more volatile molecules and produced higher volumes of nectar than female-phase flowers. Alkaloids in pollen functioned as chemical defences, and were more diverse and more concentrated compared to the alkaloids in nectar. Visitors actively collected little pollen for larval food but consumed more of the less-toxic nectar. Toxic pollen remaining on the bee bodies promoted pollen transfer efficiency, facilitating pollination.

Influence of spatial distribution and size of clones on the realized outcrossing rate of the marsh cinquefoil (Comarum palustre).

BACKGROUND AND AIMS: Clonal growth is a common feature in flowering plants. As clone size increases, the selfing rate in self-compatible species is likely to increase due to more frequent geitono-pollination events (i.e. pollination among flowers within the same genet). This study investigated the breeding system of the marsh cinquefoil (Comarum palustre) and assessed spatial distribution of clones, clone size and architecture, and their effects on realized outcrossing rates. In addition, pollen dispersal was investigated in two patchy populations. METHODS: The species' breeding system was investigated under controlled conditions through hand pollinations (self- vs. cross-pollination). Using microsatellite markers, an assessment was made of the realized outcrossing rates and the genetic diversity in four natural populations, the clonal structure in two populations within five 15 × 15 m sampling plots following 0.5 × 0.5 m grids, and the pollen dispersal through paternity assignment tests in those two populations. KEY RESULTS: Comarum palustre is a self-compatible species but only presents a low rate of spontaneous self-pollination. The occurrence of inbreeding depression was not detected at the seed set stage (δ(SS) = 0.04). Clones were spatially clumped (A(C) = 0.60-0.80), with intermediate to no intermingling of the ramets (D(C) = 0.40-1.00). Genet size ranged from one to 171 ramets. Patchy populations had low outcrossing rates (t(m) = 0.33-0.46). Large clones showed lower outcrossing rates than small clones. Pollen dispersal mainly occurred within patches as only 1-7 % of the pollination events occurred between patches of >25 m separation. Seedling recruitment events were detected. CONCLUSIONS: Genet size together with distances between patches, through increasing geitono-pollination events, appeared to be important factors influencing realized outcrossing rates. The study also revealed seed flow allowing seedling recruitment, which may contribute to increasing the number of new patches, and potentially further enhance gene flow within populations.

Development and multiplexing of microsatellite markers using pyrosequencing in the clonal plant Comarum palustre (Rosaceae).

Microsatellites represent one of the most commonly used genetic markers for population genetic studies. Traditionally, their development is quite time consuming, requiring construction of a genomic library enriched for repeated motifs. Using pyrosequencing, a fast and cost-effective new generation sequencing technique, we produced 24,340,862 bases in 63,860 short fragment reads, including 1170 dinucleotide motifs with a minimum of six repeats and 1383 trinucleotide motifs with a minimum of four repeats for the Marsh Cinquefoil, Comarum palustre L., an endangered marsh pioneer species. We selected 58 loci with SSR (Short Sequence Repeat) segments (at least 10 repeats) for a preliminary screening. Out of them, we screened 29 loci on a capillary sequencer after ligation in a vector and PCR using T7 forward primer labelled with FAM fluorescent dye and the specific unlabeled reverse primers. This procedure allowed us to screen large number of candidate loci with the same labelled primer and unlabelled specific primers. Finally, we characterized 20 polymorphic microsatellite markers, nine dinucleotides and 11 trinucleotides. We used these markers to assess genetic diversity and clonal structure in two Belgian populations. All loci showed a maximum of two alleles per individual, suggesting that they are from a diploid genome. One genet was detected in a newly extending population while 53 different genets in a long-term ecologically managed population. The number of alleles per locus ranged from 6 to 14 in this old population with an expected heterozygosity, ranging from 0.5964 to 0.8278. These preliminary results show a genet size up to 7.2 m.

Influence of clonal growth on selfing rate in Vaccinium myrtillus L.

Clonal growth, which allows the multiplication of flowering shoots of the same genet, can lead to a large floral display and may thus increase the rate of selfing through geitonogamy as a consequence of an increase in the number of successively visited flowers. The aim of the present research was to analyse the combined effect of the diversity and intermingling of clones on the rate of selfing in Vaccinium myrtillus. Four mother plants were selected within patches characterised by contrasting clonal structure (low versus high number and intermingling of clones). The selfing rate was significantly lower for plants situated within patches characterised by a high number of intermingled clones (3%) than for plants situated in patches with a low number of clones (50%). Therefore, for this species suffering from inbreeding depression, an increase in the number or the intermingling of the clones could reduce the rate and the cost of geitonogamy and allow a large floral display to attract pollinators. We also found that the main pollinators, bumblebee queens, presented a foraging behaviour favouring geitonogamy, as their successive visits to flowers were quite short (89% of flights were 40 cm or less).

Evolutionary mode, tempo, and phylogenetic association of continuous morphological traits in the aquatic moss genus Amblystegium.

Evolutionary significance of morphological characters that have traditionally been used for species delineation in the aquatic moss genus Amblystegium was tested by partitioning the environmentally and genetically induced morphological variation and focusing on morphological evolution using comparative methods. Cultivation experiments under controlled condition showed that most of the morphological variation in nature resulted from plasticity. Information regarding genetically fixed morphological variation and genetic similarity derived from polymorphic inter-simple sequence repeat markers was combined into an explicit model of morphological evolution. Maximum likelihood estimates of the model parameters indicated that evolution of most characters tended to accelerate in the most recent taxa and was often independent from the phylogeny. Constraining the different characters to be independent from each other most often produced a less likely result than when the characters were free to evolve in a correlated fashion. Thus, the morphological characters that have traditionally been used to circumscribe different Amblystegium species lack the independence, diagnostic value for specific lineages, and stability that would be required for distinguishing different species.

Chloroplast DNA haplotype variation and population differentiation in Sorbus aucuparia L. (Rosaceae: Maloideae).

Intra-specific chloroplast DNA (cpDNA) variation was studied in Sorbus aucuparia L., an entomophilous, mid-or early successional tree producing fleshy fruits. Eight PCR-amplified fragments of the chloroplast genome were screened for restriction fragment length polymorphisms, using one or two 4 bp-cutter restriction endonucleases. cpDNA variation was investigated on two geographical scales: (1) among four regions in France and Belgium; and (2) within the Belgian region. A total of 150 individuals from six populations were analysed. Fourteen polymorphisms were detected in six of the cpDNA fragments. All polymorphisms probably resulted from insertions or deletions, and allowed the identification of 12 haplotypes. The level of genetic differentiation computed on the basis of haplotype frequencies was similar on the two geographical scales considered (G(STc) = 0.286 among regions, G(STc) = 0.259 among populations within the Belgian region). These values are much lower than those obtained in nine previously studied temperate tree species, which are all wind-pollinated, late-successional species producing dry fruits. These results might primarily be accounted for by the contrasting life history traits of S. aucuparia. In order to obtain insights into the relative contribution of pollen and seeds to gene flow, G(STc) was also compared with previously obtained G(ST) estimates based on allozyme data.

Allozyme diversity and genetic structure in South-Western populations of heather, Calluna vulgaris.

Genetic diversity, population genetic structure and gene flow in Calluna vulgaris (L.) Hull were assessed by means of seven allozyme loci scored in 18 populations from the South-Western area of the species' range. Genetic diversity was lower (HT = 0.20) than reported for long-lived widespread species but was characterized by a high number of alleles per locus (5.60 at the species level) of which more than 70% were rare. More than 95% of genetic variation was found at the intrapopulation level (GST = 0.047). High levels of past gene flow were inferred, based on the allozyme data (Nm = 5.2 from GST . Nm = 10.2 from the 'private allele' method). Calluna vulgaris exhibited several geographic patterns of genetic variation. Both cluster analysis, constructed with various genetic distances and principal components analysis showed that Spanish and Pyrenean populations were clearly different from those collected in the Massif Central and Belgium. Also, a trend for decreasing genetic diversity towards Northern populations was detected. These patterns might be related to the post-glacial history of Calluna. In addition, it is shown that isolation by distance has played a role in the geographic shaping of genetic variation in this species.