Population size affected by environmental variability impacts genetics, traits, and plant performance in Trifolium montanum L.

Population size, genetic diversity, and performance have fundamental importance for ecology, evolution, and nature conservation of plant species. Despite well-studied relationships among environmental, genetic, and intraspecific trait variation (ITV), the influence of population size on these aspects is less understood. To assess the sources of population size variation, but also its impact on genetic, functional trait, and performance aspects, we conducted detailed population size estimations, assessed 23 abiotic and biotic environmental habitat factors, performed population genetic analyses using nine microsatellite markers, and recorded nine functional traits based on 260 Trifolium montanum individuals from 13 semi-dry grassland locations of Central Europe. Modern statistical analyses based on a multivariate framework (path analysis) with preselected linear regression models revealed that the variation of abiotic factors (in contrast to factors per se) almost completely, significantly explained fluctuations in population size (R 2 = .93). In general, abiotic habitat variation (heterogeneity) was not affected by habitat area. Population size significantly explained genetic diversity (N A: R 2 = .42, H o: R 2 = .67, H e: R 2 = .43, and I: R 2 = .59), inbreeding (F IS: R 2 = .35), and differentiation (G ST: R 2 = .20). We also found that iFDCV (ITV) was significantly explained by abiotic habitat heterogeneity, and to a lesser extent by genetic diversity H e (R 2 = .81). Nevertheless, habitat heterogeneity did not statistically affect genetic diversity. This may be due to the use of selectively neutral microsatellite markers, and possibly by insufficient abiotic selective pressures on habitats examined. Small T. montanum populations in nonoptimal habitats were characterized by reduced genetic and functional trait diversity, and elevated genetic inbreeding and differentiation. This indicates reduced adaptability to current and future environmental changes. The long-term survival of small populations with reduced genetic diversity and beginning inbreeding will be highly dependent on habitat protection and adequate land-use actions.

Nutrient Composition of Different Hazelnut Cultivars Grown in Germany.

Hazelnuts are rarely cultivated in Germany, although they are a valuable source for macro- and micronutrients and can thus contribute to a healthy diet. Near the present, 15 varieties were cultivated in Thuringia, Germany, as a pilot study for further research. The aim of our study was to evaluate the micro- and macronutrient composition of representative, randomly mixed samples of the 15 different hazelnut cultivars. Protein, fat, and fiber contents were determined using established methods. Fatty acids, tocopherols, minerals, trace elements, and ultra-trace elements were analyzed using gas chromatography, high-performance liquid chromatography, and inductively coupled plasma triple quadrupole mass-spectrometry, respectively. We found that the different hazelnut varieties contained valuable amounts of fat, protein, dietary fiber, minerals, trace elements, and α-tocopherol, however, in different quantities. The variations in nutrient composition were independent of growth conditions, which were identical for all hazelnut varieties. Therefore, each hazelnut cultivar has its specific nutrient profile.

Plant intraspecific functional trait variation is related to within-habitat heterogeneity and genetic diversity in Trifolium montanum L.

Intraspecific trait variation (ITV), based on available genetic diversity, is one of the major means plant populations can respond to environmental variability. The study of functional trait variation and diversity has become popular in ecological research, for example, as a proxy for plant performance influencing fitness. Up to now, it is unclear which aspects of intraspecific functional trait variation (iFDCV) can be attributed to the environment or genetics under natural conditions. Here, we examined 260 individuals from 13 locations of the rare (semi-)dry calcareous grassland species Trifolium montanum L. in terms of iFDCV, within-habitat heterogeneity, and genetic diversity. The iFDCV was assessed by measuring functional traits (releasing height, biomass, leaf area, specific leaf area, leaf dry matter content, Fv/Fm, performance index, stomatal pore surface, and stomatal pore area index). Abiotic within-habitat heterogeneity was derived from altitude, slope exposure, slope, leaf area index, soil depth, and further soil factors. Based on microsatellites, we calculated expected heterozygosity (He) because it best-explained, among other indices, iFDCV. We performed multiple linear regression models quantifying relationships among iFDCV, abiotic within-habitat heterogeneity and genetic diversity, and also between separate functional traits and abiotic within-habitat heterogeneity or genetic diversity. We found that abiotic within-habitat heterogeneity influenced iFDCV twice as strong compared to genetic diversity. Both aspects together explained 77% of variation in iFDCV ( R adj 2  = .77, F 2, 10 = 21.66, p < .001). The majority of functional traits (releasing height, biomass, specific leaf area, leaf dry matter content, Fv/Fm, and performance index) were related to abiotic habitat conditions indicating responses to environmental heterogeneity. In contrast, only morphology-related functional traits (releasing height, biomass, and leaf area) were related to genetics. Our results suggest that both within-habitat heterogeneity and genetic diversity affect iFDCV and are thus crucial to consider when aiming to understand or predict changes of plant species performance under changing environmental conditions.

Characterization of 15 nuclear microsatellite markers for Afzelia africana (Fabaceae) and related species.

PREMISE: Afzelia africana (Fabaceae) is a valuable, internationally vulnerable tree species in tropical Africa. The development of specific simple sequence repeat (SSR) loci is necessary for population genetic studies in this tree species and its closest relatives. METHODS AND RESULTS: Fifteen new polymorphic microsatellite markers were developed for A. africana using Illumina next-generation sequencing. We tested the polymorphism of the 15 loci in three populations in Benin, West Africa. The number of expressed alleles per locus varied from one to 12. The levels of observed and expected heterozygosity ranged from 0.100 to 1.000 and from 0.095 to 0.882, respectively. Most markers successfully amplified in the closely related species A. quanzensis and A. bipindensis, but less so in A. bipindensis. CONCLUSIONS: Because of their cross-amplification ability, these newly developed loci will serve as useful tools for future molecular analyses on A. africana and related species.

Genetic characterization and barcoding of taxa in the genus Wolffia Horkel ex Schleid. (Lemnaceae) as revealed by two plastidic markers and amplified fragment length polymorphism (AFLP).

The genus Wolffia of the duckweed family (Lemnaceae) contains the smallest flowering plants. Presently, 11 species are recognized and categorized mainly on the basis of morphology. Because of extreme reduction of structure of all species, molecular methods are especially required for barcoding and identification of species and clones of this genus. We applied AFLP combined with Bayesian analysis of population structure to 66 clones covering all 11 species. Nine clusters were identified: (1) W. angusta and W. microscopica (only one clone), (2) W. arrhiza, (3) W. cylindracea (except one clone that might be a transition form), (4) W. australiana, (5) W. globosa, (6) W. globosa, W. neglecta, and W. borealis, (7) W. brasiliensis, and W. columbiana, (8) W. columbiana, (9) W. elongata. Furthermore, we investigated the sequences of plastidic regions rps16 (54 clones) and rpl16 (55 clones), and identified the following species: W. angusta, W. australiana, W. brasiliensis, W. cylindracea, W. elongata, W. microscopica, and W. neglecta. Wolffia globosa has been separated into two groups by both methods. One group which consists only of clones from North America and East Asia was labelled here "typical W. globosa". The other group of W. globosa, termed operationally "W. neglecta", contains also clones of W. neglecta and shows high similarity to W. borealis. None of the methods recognized W. borealis as a distinct species. Although each clone could be characterized individually by AFLP and plastidic sequences, and most species could be bar-coded, the presently available data are not sufficient to identify all taxa of Wolffia.

Genetic structure of the genus Lemna L. (Lemnaceae) as revealed by amplified fragment length polymorphism.

Duckweeds (Lemnaceae) are extremely reduced in morphology, which made their taxonomy a challenge for a long time. The amplified fragment length polymorphism (AFLP) marker technique was applied to solve this problem. 84 clones of the genus Lemna were investigated representing all 13 accepted Lemna species. By neighbour-joining (NJ) analysis, 10 out of these 13 species were clearly recognized: L. minor, L. obscura, L. turionifera, L. japonica, L. disperma, L. aequinoctialis, L. perpusilla, L. trisulca, L. tenera, and L. minuta. However, L. valdiviana and L. yungensis could be distinguished neither by NJ cluster analysis nor by structure analysis. Moreover, the 16 analysed clones of L. gibba were assembled into four genetically differentiated groups. Only one of these groups, which includes the standard clones 7107 (G1) and 7741 (G3), represents obviously the "true" L. gibba. At least four of the clones investigated, so far considered as L. gibba (clones 8655a, 9481, 9436b, and Tra05-L), represent evidently close relatives to L. turionifera but do not form turions under any of the conditions tested. Another group of clones (6745, 6751, and 7922) corresponds to putative hybrids and may be identical with L. parodiana, a species not accepted until now because of the difficulties of delineation on morphology alone. In conclusion, AFLP analysis offers a solid base for the identification of Lemna clones, which is particularly important in view of Lemnaceae application in biomonitoring.

Removal of noisy characters from chloroplast genome-scale data suggests revision of phylogenetic placements of Amborella and Ceratophyllum.

It is widely appreciated that noisy, highly variable data can impede phylogeney reconstruction. Researchers have for a long time omitted problematic data from phylogenetic analyses, such as the third-codon positions and variable regions. In the analyses of the phylogenetic relations of the angiosperms; however, inclusion of complete gene sequences into genomic-scale alignments has become a common practice. Here we demonstrate that this practice can be misleading. We show that support of the basal-most position of Amborella trichopoda among the angiosperms in the chloroplast genomic data is based only on a tiny subset (< 1% of the total alignment length) of the most variable positions in alignment, exhibiting mean maximum likelihood (ML) distance among the angiosperm operational taxonomic units (OTUs) approximately 36 substitutions/site. Exclusion of these positions leads to disappearance of the basal Amborella branch. Likewise, the recently reported sister-group relationship of Ceratophyllum to the eudicots is based on the presence of 2% of the most variable positions in the genomic alignment, exhibiting, on average, 20 substitutions/site in comparison among the angiosperm OTUs. These observations highlight a need for excluding a certain proportion of saturated positions in alignment from phylogenomic analyses.

The molecular phylogeny of Rebutia (Cactaceae) and its allies demonstrates the influence of paleogeography on the evolution of South American mountain cacti.

The tropical Andes harbor a major part of the world's plant biodiversity. The montane cacti of the tribes Browningieae, Cereeae, and Trichocereeae underwent extensive radiation and thus are well suited as a model group to study the diversification of Andean plants. We reconstructed their phylogeny employing three noncoding chloroplast regions and explained it in the context of the geological history of South America. We found that the clade of cephalia-bearing cacti with naked pericarpels is centered in northeastern Brazil, whereas almost all other clades comprise Andean species. The spatial split between the clades was probably caused by the Andean uplift and the concurrent formation of intracontinental marine basins in the Tertiary. The phylogenetic reconstructions based on parsimony and Bayesian approaches do not reflect the traditional delimitation of the tribes and of the large genera. Our results suggest that Rebutia s.l. and Echinopsis s.l. are not monophyletic and that Sulcorebutia, Weingartia, and Cintia should be united into one genus. Even though this "Weingartia-complex" and the genus Gymnocalycium are similar in size and morphological diversity, Gymnocalycium has a very high molecular divergence suggesting a comparably older radiation.

A new test of phylogenetic model fitness addresses the issue of the basal angiosperm phylogeny.

We readdress the issue of phylogeny of the basal extant angiosperms employing a source previously not systematically investigated, specifically, the non-coding sequences of cpDNA. Comparison of trees with and without grasses or the outgroup (Pinus) in our analyses revealed no rearrangements in tree topology that might be expected if LBA were distorting the position of the magnoliids. For each model applied, irrespective of whether monocots or ANITA members appeared basally divergent, the orchid Phalaenopsis assumed the same position on the trees with the reduced taxon set as did the branch bearing the orchid plus the grasses in the full alignment. However, our new test of model fitness revealed a different flaw influencing the placement of monocots, which is related to model mis-specification. This flaw similarly affects the full alignment and the alignment with grasses removed. In both cases the models favouring a relatively derived position for the monocots and basal placement of the branch of Amborella plus Nymphaea provide better overall prediction of the observed data structure. In the view of apparent unsuitability of the bootstrap method for large data sets, our novel test provides a new means of exploring conflicts caused by systematic errors in phylogenetic analyses.

Analysis of Acorus calamus chloroplast genome and its phylogenetic implications.

Determining the phylogenetic relationships among the major lines of angiosperms is a long-standing problem, yet the uncertainty as to the phylogenetic affinity of these lines persists. While a number of studies have suggested that the ANITA (Amborella-Nymphaeales-Illiciales-Trimeniales-Aristolochiales) grade is basal within angiosperms, studies of complete chloroplast genome sequences also suggested an alternative tree, wherein the line leading to the grasses branches first among the angiosperms. To improve taxon sampling in the existing chloroplast genome data, we sequenced the chloroplast genome of the monocot Acorus calamus. We generated a concatenated alignment (89,436 positions for 15 taxa), encompassing almost all sequences usable for phylogeny reconstruction within spermatophytes. The data still contain support for both the ANITA-basal and grasses-basal hypotheses. Using simulations we can show that were the ANITA-basal hypothesis true, parsimony (and distance-based methods with many models) would be expected to fail to recover it. The self-evident explanation for this failure appears to be a long-branch attraction (LBA) between the clade of grasses and the out-group. However, this LBA cannot explain the discrepancies observed between tree topology recovered using the maximum likelihood (ML) method and the topologies recovered using the parsimony and distance-based methods when grasses are deleted. Furthermore, the fact that neither maximum parsimony nor distance methods consistently recover the ML tree, when according to the simulations they would be expected to, when the out-group (Pinus) is deleted, suggests that either the generating tree is not correct or the best symmetric model is misspecified (or both). We demonstrate that the tree recovered under ML is extremely sensitive to model specification and that the best symmetric model is misspecified. Hence, we remain agnostic regarding phylogenetic relationships among basal angiosperm lineages.

The chloroplast genome of Nymphaea alba: whole-genome analyses and the problem of identifying the most basal angiosperm.

Angiosperms (flowering plants) dominate contemporary terrestrial flora with roughly 250,000 species, but their origin and early evolution are still poorly understood. In recent years, molecular evidence has accumulated suggesting a dicotyledonous origin of monocots. Phylogenetic reconstructions have suggested that several dicotyledonous groups that include taxa such as Amborella, Austrobaileya, and Nymphaea branch off as the most basal among angiosperms. This has led to the concept of monocots, "eudicots," "basal dicots," and "ANITA" groupings. Here, we present the sequence and phylogenetic analyses of the chloroplast DNA of Nymphaea alba. Phylogenetic analyses of our 14-species data set, consisting of 29,991 aligned nucleotide positions per chloroplast genome, revealed consistent support for Nymphaea being a divergent member of a monophyletic dicot assemblage. Three distinct angiosperm lineages were supported in the majority of our phylogenetic analyses-eudicots, Magnoliopsida, and monocots. However, the monocot lineage leading to the grasses was the deepest branching. Although analyses of only one individual gene alignment (out of 61) is consistent with some recently proposed hypotheses for the paraphyly of dicots, we also report observations that nine genes do not support paraphyly of dicots. Instead, they support the basal monocot-dicot split. Consistent with this finding, we also report observations suggesting that the monocot lineage leading to the grasses has the strongest phylogenetic affinity to gymnosperms. Our findings have general implications for studies of substitution model specification and analyses of concatenated genome data.

Genetic diversity of Ranunculus acris L. (Ranunculaceae) populations in relation to species diversity and habitat type in grassland communities.

Correlates between genetic diversity at intra- and interpopulation levels and the species diversity in plant communities are rarely investigated. Such correlates may give insights into the effect of local selective forces across different communities on the genetic diversity of local plant populations. This study has employed amplified fragment length polymorphism to assess the genetic diversity within and between 10 populations of Ranunculus acris in relation to the species diversity (richness and evenness) of grassland communities of two different habitat types, 'seminatural' and 'agriculturally improved', located in central Germany. Within-population genetic diversity estimated by Nei's unbiased gene diversity (HE) was high (0.258-0.334), and was not correlated with species richness (Pearson's r = -0.17; P = 0.64) or species evenness (Pearson's r = 0.15; P = 0.68) of the plant communities. However, the genetic differentiation between R. acris populations was significantly correlated with the difference in species evenness (Mantel's r = 0.62, P = 0.02), but not with difference in species richness of plant communities (r = -0.17, P = 0.22). Moreover, we also found that populations of R. acris from the 'seminatural' habitat were genetically different (amova, P < 0.05) from those in 'agriculturally improved' habitats, suggesting that gene flow between these habitat types is limited. The results reported in this study may indicate that habitat characteristics influence the genetic diversity of plant species.

Analysis of the Amborella trichopoda chloroplast genome sequence suggests that amborella is not a basal angiosperm.

Phylogenetic analyses based on comparison of a limited number of genes recently suggested that Amborella trichopoda is the most ancient angiosperm. Here we present the complete sequence of the chloroplast genome of this plant. It does not display any of the genes characteristic of chloroplast DNA of the gymnosperm Pinus thunbergii (chlB, chlL, chlN, psaM, and ycf12). The majority of phylogenetic analyses of protein-coding genes of this chloroplast DNA suggests that Amborella is not the basal angiosperm and not even the most basal among dicots.