Study in Agaricus subgenus Minores and allied clades reveals a new American subgenus and contrasting phylogenetic patterns in Europe and Greater Mekong Subregion.

Within Agaricus subg. Minores, A. sect. Minores remains a little-studied section due generally to its delicate sporocarps often lacking taxonomically relevant morphological characters. To reconstruct the section, using the recent taxonomic system based on divergence times, and to evaluate the species diversity of A. sect. Minores in the Greater Mekong Subregion, 165 specimens were incorporated in phylogenetic analyses. A dated tree based on nuclear ITS, LSU and tef1-α sequence data allowed us to better circumscribe A. subg. Minores and to propose a new subgenus, A. subg. Minoriopsis, which is only known from tropical and subtropical regions of the Americas. A larger tree based on ITS sequences indicated that, with 81 phylogenetic species, the reconstructed section Minores is now one of the largest sections in the genus. Within A. subg. Minores, a new section, A. sect. Leucocarpi, and eleven new species are described from the Greater Mekong Subregion. Thirty-eight species of A. sect. Minores from this region of Asia were distributed in multiple clades that successively diverged over the past 24 million years. In contrast, species reported from Europe mostly grouped in a single non-tropical clade, suggesting a major species diversification following the middle Miocene climatic optimum.

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.

Two reproductively isolated cytotypes and a swarm of highly inbred, disconnected populations: a glimpse into Salicornia's evolutionary history and challenging taxonomy.

The main factor of differentiation at six nuclear microsatellite and seven cpDNA loci in Salicornia from the Mediterranean and Atlantic coasts of France is cytotypic identity, suggesting the presence of a strong reproductive barrier among sympatric cytotypes. Within cytotypes, a substantial proportion of the differentiation between species is due to confounded phylogeographic signal. Conspecific individuals tend to be significantly more related than individuals from different species at the population scale, but mean kinship coefficients among pairs of conspecific and nonconspecific individuals from different populations are not significantly different, suggesting the absence of reproductive isolation among species of the same cytotype. The observed association between morphology and genetic variation within populations would thus result from the selfing mating system (F(is)) = 0.70) generating substantial linkage within the genome, linkage that would quickly disappear among unrelated individuals from different populations. Salicornia species thus function as a network of inbred populations, strongly challenging taxonomic concepts.

Fitness and genetic variation of Viola calaminaria, an endemic metallophyte: implications of population structure and history.

We investigated variations in genetic diversity and plant fitness in a rare endemic metallophyte of calamine soils, Viola calaminaria, in relation to population size, population connectivity and population history in order to evaluate and discuss potential conservation strategies for the species. Mean population genetic diversity (H(s) = 0.25) of V. calaminaria was similar to endemic non-metallophyte taxa. Twenty-one per cent of the genetic variation was partitioned among populations and a low (9%) but significant differentiation was found among geographical regions. Our results did not support the hypothesis that the acquisition of metal tolerance may result in reduced genetic diversity, and suggested that strict metallophytes do not exhibit higher inter-population differentiation resulting from scattered habitats. There were no relationships between population genetic diversity and population size. Significant correlations were found between plant fitness and (i) population size and (ii) connectivity index. Recently-founded populations exhibited the same level of genetic diversity as ancient populations and also possessed higher plant fitness. There was no indication of strong founder effects in recently-established populations. The results suggest that the creation of habitats through human activities could provide new opportunities for conservation of this species.

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).

S-allele diversity in Sorbus aucuparia and Crataegus monogyna (Rosaceae: Maloideae).

RT-PCR was used to obtain the first estimates from natural populations of allelic diversity at the RNase-based gametophytic self-incompatibility locus in the Rosaceae. A total of 20 alleles were retrieved from 20 Sorbus aucuparia individuals, whereas 17 alleles were found in 13 Crataegus monogyna samples. Estimates of population-level allele numbers fall within the range observed in the Solanaceae, the only other family with RNase-based incompatibility for which estimates are available. The nucleotide diversity of S-allele sequences was found to be much lower in the two Rosaceae species as compared with the Solanaceae. This was not due to a lower sequence divergence among most closely related alleles. Rather, it is the depth of the entire genealogy that differs markedly in the two families, with Rosaceae S-alleles exhibiting more recent apparent coalescence. We also investigated patterns of selection at the molecular level by comparing nucleotide diversity at synonymous and nonsynonymous sites. Stabilizing selection was inferred for the 5' region of the molecule, while evidence of diversifying selection was present elsewhere.

Inheritance of the chloroplast genome in Sorbus aucuparia L. (Rosaceae).

Chloroplast DNA (cpDNA) inheritance was investigated in Sorbus aucuparia using progenies obtained from six controlled crosses between individuals of known haplotype. Polymerase chain reaction (PCR) amplification, followed by restriction analysis, was used to characterize 248 offspring for either of two polymorphic cpDNA fragments. All offspring exhibited the maternal haplotype, which indicates maternal inheritance of chloroplasts in S. aucuparia. Power analysis of the test showed that the frequency of paternal transmission of chloroplasts, if any, should not exceed 1.84% (with 99% confidence).

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.