Supplementary Table E.1

Characterisation of loci by MCHEZA, BayeScan and Samβada. For each locus, estimates of F_ST and associated significance values obtained using MCHEZA and BayeScan are given. Outlier loci found by Samβada are documented together with the environmental variable(s) showing significant correlations with the presence of the dominant allele ordered according to descending Wald scores. Additionally, the allele frequency differentials (δ) along with estimates of the genomic clines analysis as calculated by Introgress are shown. ‘Genomic cline’ indicates whether a locus shows a significant excess (H+) or deficit (H-) of S. hercynicus genotypes in a S. ovatus background. Fisher’s exact test statistics (χ², P_adj) are given, indicating whether a locus was found as being taxon-specific (H - S. hercynicus; O - S. ovatus). P-values (P_adj) were adjusted according to Benjamini and Hochberg (1995) to account for multiple testing. The last column gives the Sperman's rank correlation coefficient and P-value for the correlations tested between environmental variables detected by Samβada and frequencies of the present allele (band presence). dark red - loci that were found as being under divergent selection, that showed a significant relationship with an environmental variable, that showed significant deviation from a model of neutral introgression and that were found as being taxon-specific; light red - loci with allele frequency differentials δ > 0.25

Hybridisation and its consequences: Population genomics, herbivory, and phytochemistry in the Senecio nemorensis syngameon

Hybridisation often determines the evolutionary trajectories of closely related species and may lead on the one hand to the formation of new species or on the other hand to a loss of species diversity through genetic swamping or fusion of taxa. The three Central European representatives of the Senecio nemorensis syngameon, S. germanicus, S. ovatus, and S. hercynicus are vertically vicariant species that hybridise in their range overlaps. In the present dissertation the following three key aspects concerning consequences of hybridisation among the three species are highlighted. Firstly, by using a population genomic approach, the extent of hybridisation between S. hercynicus and S. ovatus and possible natural selection regimes are investigated along four elevational transects in the Bavarian Forest National Park. Secondly, transplantation experiments were performed to test for differences in consumption of pure species and their artificial F1 hybrids by herbivores along an elevational gradient. Finally, in food choice experiments the observed results from the field were checked under controlled lab conditions and a possible relationship between consumption and chemical defence compounds (i.e., pyrrolizidine alkaloids and tannins) was investigated. Addressing the first key aspect, we found advanced introgressive hybridisation along the whole transects. Most individuals could be assigned to the S. ovatus backcross class and just very few S. hercynicus-like genotypes were present. Although evidence was found for a climate-mediated divergent selection, this force is interpreted as not being strong enough to maintain the two biological species. During the transplantation experiment it was shown that S. hercynicus also suffers to a higher extent from herbivory than S. ovatus but there was also evidence for an elevated susceptibility to low temperatures of the latter at elevations typical for S. hercynicus. In contrast, S. germanicus showed low herbivore damage relative to S. hercynicus and low leaf area loss through withering relative to S. ovatus. In the food choice experiments, the results from the field could be proven to some extent. It was shown that as soon as four leaf discs of the different taxa (i.e., parental species and reciprocal hybrids) were offered to the molluscs, the selective consumption found for the pure taxa was compensated. Hybrids were neither more susceptible nor more resistant than their corresponding parental species. A correlation of feeding preference and content of chemical defence compounds could only partly be proven for pyrrolizidine alkaloids. Tannins had no effect at all on the consumption by molluscs. To conclude, all results from the present studies indicate that S. hercynicus will lose to S. ovatus in the Bavarian Forest and neither climate- nor herbivory-mediated selection seems strong enough to keep both species as divergent lineages. In the S. germanicus-S. ovatus system the evolutionary trajectories are not assumed to lead to an extinction of either species. Rather, a patchily distribution of pure and hybrid populations is expected from the results

Duckweed (Lemnaceae): Its Molecular Taxonomy

Duckweeds include the world's smallest and fastest growing flowering plants that have the capacity to produce huge biomass with a broad range of potential applications like production of feed and food, biofuel and biogas. In order to achieve optimal and sustainable commercial system, it is necessary that suitable species and clones of duckweeds be identified and selected based on appropriate strategies. However, a high degree of reduction in their structural complexity poses serious problems in identification of closely related species of duckweeds, on a morphological basis. Use of molecular taxonomic tools is the present solution. The state of the art of molecular taxonomy of all the five genera of duckweeds (Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia) is based mainly on the techniques of fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding using sequences of plastidic DNA fragments. After more than 15 years of molecular taxonomic investigations, a certain viewpoint is now available demonstrating all five genera to be monophyletic. Also, the phenetic analyses had made huge progress in delineating the currently defined 36 species of duckweeds, although, all species cannot yet be defined with confidence. Wolffiella has turned out to be the most complicated genus as only 6 to 7 species out of the 10 can be reliably delineated. Further progress in the phylogenetic and phenetic analyses requires more advanced methods like next generation and/or whole genome sequencing. First results using the method genotyping-by-sequencing in the genus Lemna (in combination with metabolomic profiling by matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) as well as AFLP and barcoding by plastidic sequences) are more promising: The species Lemna valdiviana and Lemna yungensis were united to one species, Lemna valdiviana. This reduced the total number of Lemnaceae species to 36

Are there morphological and life-history traits under climate-dependent differential selection in S Tunesian Diplotaxis harra (Forssk.) Boiss. (Brassicaceae) populations?

Adaptation of morphological, physiological, or life-history traits of a plant species to heterogeneous habitats through the process of natural selection is a paramount process in evolutionary biology. We have used a population genomic approach to disentangle selection-based and demography-based variation in morphological and life-history traits in the crucifer Diplotaxis harra (Forssk.) Boiss. (Brassicaceae) encountered in populations along aridity gradients in S Tunisia. We have genotyped 182 individuals from 12 populations of the species ranging from coastal to semidesert habitats using amplified fragment length polymorphism (AFLP) fingerprinting and assessed a range of morphological and life-history traits from their progeny cultivated under common-garden conditions. Application of three different statistical approaches for searching AFLP loci under selection allowed us to characterize candidate loci, for which their association with the traits assessed was tested for statistical significance and correlation with climate data. As a key result of this study, we find that only the shape of cauline leaves seems to be under differential selection along the aridity gradient in S Tunisian populations of Diplotaxis harra, while for all other traits studied neutral biogeographical and/or random factors could not be excluded as explanation for the variation observed. The counter-intuitive finding that plants from populations with more arid habitats produce broader leaves under optimal conditions of cultivation than those from more mesic habitats is interpreted as being ascribable to selection for a higher plasticity in this trait under more unpredictable semidesert conditions compared to the more predictable ones in coastal habitats

Effect of the growth medium composition on nitrate accumulation in the novel protein crop Lemna minor

Duckweed is a potential alternative protein source for food and feed. However, little is known about the nitrate accumulation in this plant. A high nitrate level in vegetables can indirectly lead to an elevated intake of nitrites and N-nitroso compounds, increasing the risk of diseases for humans and animals. This research hypothesizes that the nitrate accumulation of Lemna minor differs between growing media. Additionally, it evaluates whether legal safety levels of nitrate for human and animal intake are exceeded. The duckweed was grown on (i) rainwater, and (ii) three synthetic media containing different nutrient levels. Furthermore, (iii) biological effluent of swine manure treatment and (iv) aquaculture effluent from pikeperch production were used, as these are potential media for closing nutrient loops in the agriculture sector. It was found that nitrate levels increased with the increasing availability of macronutrients in the water, and pH showed a particularly strong negative correlation with the nitrate levels in the plant. Nevertheless, nitrate content never exceeded 530 mg NO3 kg−1 fresh weight. To conclude, Lemna minor's nitrate content was below safety limits for human consumption in all tested growing media; however, a potential risk for ruminants was observed as these are more sensitive to nitrate conversions in their gastro-intestinal track

Lemnaceae and Orontiaceae Are Phylogenetically and Morphologically Distinct from Araceae

Duckweeds comprise a distinctive clade of pleustophytic monocots that traditionally has been classified as the family Lemnaceae. However, molecular evidence has called into question their phylogenetic independence, with some authors asserting instead that duckweeds should be reclassified as subfamily Lemnoideae of an expanded family Araceae. Although a close phylogenetic relationship of duckweeds with traditional Araceae has been supported by multiple studies, the taxonomic disposition of duckweeds must be evaluated more critically to promote nomenclatural stability and utility. Subsuming duckweeds as a morphologically incongruent lineage of Araceae effectively eliminates the family category of Lemnaceae that has been widely used for many years. Instead, we suggest that Araceae subfamily Orontioideae should be restored to family status as Orontiaceae, which thereby would enable the recognition of three morphologically and phylogenetically distinct lineages: Araceae, Lemnaceae, and Orontiaceae

Nutritional Value of the Duckweed Species of the Genus Wolffia (Lemnaceae) as Human Food

Species of the genus Wolffia are traditionally used as human food in some of the Asian countries. Therefore, all 11 species of this genus, identified by molecular barcoding, were investigated for ingredients relevant to human nutrition. The total protein content varied between 20 and 30% of the freeze-dry weight, the starch content between 10 and 20%, the fat content between 1 and 5%, and the fiber content was ~25%. The essential amino acid content was higher or close to the requirements of preschool-aged children according to standards of the World Health Organization. The fat content was low, but the fraction of polyunsaturated fatty acids was above 60% of total fat and the content of n-3 polyunsaturated fatty acids was higher than that of n-6 polyunsaturated fatty acids in most species. The content of macro- and microelements (minerals) not only depended on the cultivation conditions but also on the genetic background of the species. This holds true also for the content of tocopherols, several carotenoids and phytosterols in different species and even intraspecific, clonal differences were detected in Wolffia globosa and Wolffia arrhiza. Thus, the selection of suitable clones for further applications is important. Due to the very fast growth and the highest yield in most of the nutrients, Wolffia microscopica has a high potential for practical applications in human nutrition

Genetic structure of the protist Physarum albescens (Amoebozoa) revealed by multiple markers and genotyping by sequencing

Myxomycetes are terrestrial protists with many presumably cosmopolitan species dispersing via airborne spores. A truly cosmopolitan species would suffer from outbreeding depression hampering local adaptation, while locally adapted species with limited distribution would be at a higher risk of extinction in changing environments. Here, we investigate intraspecific genetic diversity and phylogeography of Physarum albescens over the entire Northern Hemisphere. We sequenced 324 field collections of fruit bodies for 1-3 genetic markers (SSU, EF1A, COI) and analysed 98 specimens with genotyping by sequencing. The structure of the three-gene phylogeny, SNP-based phylogeny, phylogenetic networks, and the observed recombination pattern of three independently inherited gene markers can be best explained by the presence of at least 18 reproductively isolated groups, which can be seen as cryptic species. In all intensively sampled regions and in many localities, members of several phylogroups coexisted. Some phylogroups were found to be abundant in only one region and completely absent in other well-studied regions, and thus may represent regional endemics. Our results demonstrate that the widely distributed myxomycete species Ph. albescens represents a complex of at least 18 cryptic species, and some of these seem to have a limited geographical distribution. In addition, the presence of groups of presumably clonal specimens suggests that sexual and asexual reproduction coexist in natural populations of myxomycetes

Herbivory and fitness components in an introgressive hybrid swarm of Senecio hercynicus and S. ovatus (Compositae, Senecioneae)

Hybridization among plant species may have considerable evolutionary consequences, from the formation of new taxa to the extinction of species by genetic swamping. The evolutionary fate of a system of hybridizing species is highly dependent on fitness differences between parental and hybrid genotypes. In an introgressed hybrid swarm of the rare Senecio hercynicus and its widely distributed congener Senecio ovatus (Compositae, Senecioneae) we assessed pollen fertility, seed set, and germination rates, along with leaf damage dynamics due to herbivory throughout the vegetation period of 2006 in order to compare intrinsic and extrinsic fitness components among genotypes. Correlation and linear regression analyses carried out with fitness components as dependent and genetic constitution, phenology, and pyrrolizidine alkaloid (PA) content as independent variables reveal that: (a) intrinsic fitness components (pollen fertility, seed set, germination rate) are not reduced in hybrid and backcross individuals, (b) the extrinsic fitness component of leaf damage caused by herbivores is significantly influenced by genetic constitution (S. hercynicus-like individuals being more severely damaged than hybrid genotypes and S. ovatus-like individuals), by phenology (early flowering individuals exhibiting higher damage than late-flowering ones), and PA concentration (with the astonishing result of hercynicus-like genotypes being increasingly damaged by herbivory with increasing PA concentrations and a reversed trend - decreasing damage with increasing PA content - in the hybrid and the ovatus-like genotypes). When all surveyed fitness parameters are considered in conjunction, the present results nicely demonstrate that hybrid genotypes neither suffer from intrinsic nor from extrinsic fitness reductions and that the rare and early-flowering S. hercynicus is outperformed by hybrids and S. ovatus-like genotypes. The present study, therefore, adds further support to the prediction that S. hercynicus will be genetically swamped by the spreading S. ovatus. (C) 2016 Elsevier GmbH. All rights reserved

Lost in the hybridisation vortex: high-elevation Senecio hercynicus (Compositae, Senecioneae) is genetically swamped by its congener S. ovatus in the Bavarian Forest National Park (SE Germany)

Hybridisation is an important evolutionary process. The investigation of hybridisation along elevational gradients, with their steep abiotic and biotic clines, provides insight into the adaptation and maintenance of species in adjacent habitats. The rare Senecio hercynicus and its spreading congener S. ovatus are vertically vicariant species that show hybridisation in their range overlaps. In the present study, we used AFLP fingerprinting of 689 individuals from 38 populations to analyse population structure and introgression patterns along four elevational transects (650-1350 m) in the Bavarian Forest National Park, Gemany. Subsequently, we searched for loci putatively under divergent selection that may maintain 'pure' species despite hybrid formation by identifying taxon-specific alleles. A maximum-likelihood based hybrid index shows that the overall genetic differentiation among all populations was very low with a vanishingly small number of pure parental individuals. Almost 75% of the investigated individuals were classified as backcrosses towards S. ovatus. The highest S. hercynicus ancestry was found in the uppermost populations of two transects. Further, we found seven taxon-specific alleles being under divergent selection that correlated with climatic variables and deviating from neutral introgression. According to our results, hybridisation of S. ovatus and S. hercynicus has reached an advanced state of genetic swamping and there seems to be no driving force that is strong enough to keep both species as different lineages. Rather, S. ovatus appears to benefit through putatively adaptive introgression