Phenotypic changes conferred by polyploidisation likely alter the ecological niche of polyploids, coming along with differences in performance compared to their diploid ancestors. However, it is largely unknown, whether these performance differences remain constant during the life history of plants. Diploid S. noricus, tetraploid S. disjunctus and hexaploid S. carniolicus s. str. of the autopolyploid species-complex of Senecio carniolicus (Asteraceae) from the easternmost Alps were reciprocally transplanted to address the following questions: Are there differences in species performances indicating niche differentiation? If so, does the resident or the higher-ploid species – which is taller-growing and possibly more tolerant against challenging abiotic and biotic conditions – show higher performance? Are performance differences consistent between early and late life history stages? Our data suggest that the hierarchy of species performance depends on the life-history stage. For early life history stages (seeds, seedlings) we mainly observed superior performance of S. carniolicus s. str. even on resident sites of the two other species. In contrast, vital rates of adults were predominantly highest for the resident species. The resident site of S. carniolicus s. str., which was expected to be environmentally least stressful (i.e. longest growing period, least affected by frost) turned out to be the most selective one, with high mortality and (nearly) no reproduction of the foreign species most likely due to top soil desiccation. Our study illustrates that there may be no clear-cut answer to the question if the resident or the higher-ploid species shows superior performance in polyploid complexes, but rather suggests that relative performance may depend on life-history stage.

Temperate mountain ranges such as the European Alps have been strongly affected by the Pleistocene glaciations. Glacial advances forced biota into refugia, which were situated either at the periphery of mountain ranges or in their interior. Whereas in the Alps peripheral refugia have been repeatedly and congruently identified, support for the latter scenario, termed “nunatak hypothesis”, is still limited and no general pattern is recognizable yet. Here, we test the hypothesis of nunatak survival for species growing in the high-alpine to subnival zones on siliceous substrate using the cushion plant Androsace alpina (Primulaceae), endemic to the European Alps, as our model species. To this end, we analysed AFLP and plastid DNA sequence data obtained from a dense and range-wide sampling. Both AFLPs and plastid sequence data identified the southwestern-most population as the most divergent one. AFLP data did not allow for discrimination of interior and peripheral populations, but rather identified two to three longitudinally separated major gene pools. In contrast, in the eastern half of the Alps several plastid haplotypes of regional or local distribution in interior ranges – the Alpine periphery mostly harboured a widespread haplotype – were indicative for the presence of interior refugia. Together with evidence from other Alpine plant species this study shows that in the eastern Alps silicicolous species of open habitats in the alpine and subnival zone survived, also or exclusively so, in interior refugia. As the corresponding genetic structure may be lost in mostly nuclear-derived, rapidly homogenising marker systems such as AFLPs or RAD sequencing tags, markers not prone to homogenization, as is the case for plastid sequences (Sanger-sequenced or extracted from an NGS data set) will continue to be important for detecting older, biogeographically relevant patterns.

Endemism in mountain ranges is considered to be the result of a number of factors, including restriction to refugia during Pleistocene climate fluctuations. However, isolation in glacial refugia cannot explain the origin of narrowly endemic taxa restricted to formerly heavily glaciated areas. Here, we investigate the phylogeny of two narrowly endemic species, Euphrasia inopinata and E. sinuata (Orobanchaceae), found exclusively in formerly heavily glaciated areas of the eastern European Alps. As both species are diploid and very similar to the widespread (allo)polyploid E. minima, we test whether the restricted distributions of E. inopinata and E. sinuata are relictual, i.e., the two species are ancestral diploid remnants of a polyploid complex, or whether they are derived, i.e., the two species are peripheral segregates of a more widespread diploid. Based on ITS sequence and AFLP fingerprint data it is shown that E. inopinata and E. sinuata, whose diploid ploidy level is confirmed for all analysed individuals via flow cytometry, are phylogenetically closely related to diploid E. alpina s. l. (series Alpinae) instead of E. minima (series Parviflorae). In addition, there is no evidence that these two diploid species participated in the formation of allotetraploid E. minima. Thus, E. inopinata and E. sinuata are interpreted as peripheral segregates of the widespread E. alpina s. l.. Shifts in pollination system from allogamy in E. alpina s. l. to autogamy in E. inopinata and E. sinuata, genetic drift in small populations, and geographic isolation at the periphery of the range of E. alpina s. str. probably contributed to the morphological and ecological differentiation of E. inopinata and E. sinuata.

Phylogenomic approaches, employing next-generation sequencing (NGS) techniques, have revolutionized systematic and evolutionary biology. Target enrichment is an efficient and cost-effective method in phylogenomics and is becoming increasingly popular. Depending on availability and quality of reference data as well as on biological features of the study system, (semi-)automated identification of suitable markers will require specific bioinformatic pipelines. Here, we established a highly flexible bioinformatic pipeline, BaitsFinder, to identify putative orthologous single copy genes (SCGs) and to construct bait sequences in a single workflow. Additionally, this pipeline has been constructed to be able to cope with challenging data sets, such as the nutritionally heterogeneous plant family Orobanchaceae. To this end, we used transcriptome data of differing quality available for four Orobanchaceae species and, as reference, SCG data from monkeyflower (Erythranthe guttata, syn. Mimulus g.; 1,915 genes) and tomato (Solanum lycopersicum; 391 genes). Depending on whether gaps were permitted in initial blast searches of the four Orobanchaceae species against the reference, our pipeline identified 1,307 and 981 SCGs with average length of 994 bp and 775 bp, respectively. Automated bait sequence construction (using 2× tiling) resulted in 38,170 and 21,856 bait sequences, respectively. In comparison to the recently published MarkerMiner 1.0 pipeline BaitsFinder identified about 1.6 times as many SCGs (of at least 900 bp length). Skipping steps specific to analyses of Orobanchaceae, BaitsFinder was successfully used in a group of non-parasitic plants (three Asteraceae species and, as reference, SCG data from Arabidopsis thaliana based on previously compiled SCGs). Thus, BaitsFinder is expected to be broadly applicable in groups, where only transcriptomes or partial genome data of differing quality are available.

Allopolyploidy has played an important role in the evolution of the flowering plants. Genome mergers are often accompanied by significant and rapid alterations of genome size and structure via chromosomal rearrangements and altered dynamics of tandem and dispersed repetitive DNA families. Recent developments in sequencing technologies and bioinformatic methods allow for a comprehensive investigation of the repetitive component of plant genomes. Interpretation of evolutionary dynamics following allopolyploidization requires both the knowledge of parentage and the age of origin of an allopolyploid. Whereas parentage is typically inferred from cytogenetic and phylogenetic data, age inference is hampered by the reticulate nature of the phylogenetic relationships. Treating subgenomes of allopolyploids as if they belonged to different species (i.e., no recombination among subgenomes) and applying cross-bracing (i.e., putting a constraint on the age difference of nodes pertaining to the same event), we can infer the age of allopolyploids within the framework of the multi-species coalescent within BEAST2. Together with a comprehensive characterization of the repetitive DNA fraction using the RepeatExplorer pipeline, we apply the dating approach in a group of closely related allopolyploids and their progenitor species in the plant genus Melampodium (Asteraceae). We dated the origin of both the allotetraploid, M. strigosum, and its two allohexaploid derivatives, M. pringlei and M. sericeum, which share both parentage and the direction of the cross, to the Pleistocene (less than 1.4 Ma). Thus, Pleistocene climatic fluctuations may have triggered formation of allopolyploids possibly in short intervals, contributing to difficulties in inferring the precise temporal order of allopolyploid species divergence of M. sericeum and M. pringlei. The relatively recent origin of the allopolyploids likely played a role in the near-absence of major changes in the repetitive fraction of the polyploids’ genomes. The repetitive elements most affected by the post-polyploidization changes represented retrotransposons of the Ty1-copia lineage Maximus and, to a lesser extent, also Athila elements of Ty3-gypsy family.

Chromosome number change (polyploidy and dysploidy) plays an important role in plant diversification and speciation. Investigating chromosome number evolution commonly entails ancestral state reconstruction performed within a phylogenetic framework, which is, however, prone to uncertainty, whose effects on evolutionary inferences are insufficiently understood. Using the chromosomally diverse plant genus Melampodium (Asteraceae) as model group, we assess the impact of reconstruction method (maximum parsimony, maximum likelihood, Bayesian methods), branch length model (phylograms versus chronograms) and phylogenetic uncertainty (topological and branch length uncertainty) on the inference of chromosome number evolution. We also address the suitability of the maximum clade credibility (MCC) tree as single representative topology for chromosome number reconstruction. Each of the listed factors causes considerable incongruence among chromosome number reconstructions. Discrepancies between inferences on the MCC tree from those made by integrating over a set of trees are moderate for ancestral chromosome numbers, but severe for the difference of chromosome gains and losses, a measure of the directionality of dysploidy. Therefore, reliance on single trees, such as the MCC tree, is strongly discouraged and model averaging, taking both phylogenetic and model uncertainty into account, is recommended. For studying chromosome number evolution, dedicated models implemented in the program ChromEvol and ordered maximum parsimony may be most appropriate. Chromosome number evolution in Melampodium follows a pattern of bidirectional dysploidy (starting from x = 11 to x = 9 and x = 14, respectively) with no prevailing direction.

Because novel environmental conditions alter the selection pressure on genes or entire subgenomes, adaptive and nonadaptive changes will leave a measurable signature in the genomes, shaping their molecular evolution. We present herein a model of the trajectory of plastid genome evolution under progressively relaxed functional constraints during the transition from autotrophy to a nonphotosynthetic parasitic lifestyle. We show that relaxed purifying selection in all plastid genes is linked to obligate parasitism, characterized by the parasite’s dependence on a host to fulfill its life cycle, rather than the loss of photosynthesis. Evolutionary rates and selection pressure coevolve with macrostructural and microstructural changes, the extent of functional reduction, and the establishment of the obligate parasitic lifestyle. Inferred bursts of gene losses coincide with periods of relaxed selection, which are followed by phases of intensified selection and rate deceleration in the retained functional complexes. Our findings suggest that the transition to obligate parasitism relaxes functional constraints on plastid genes in a stepwise manner. During the functional reduction process, the elevation of evolutionary rates reaches several new rate equilibria, possibly relating to the modified protein turnover rates in heterotrophic plastids.

Despite its evolutionary and ecological relevance the mode of polyploid origin has been notoriously difficult to be reconstructed from molecular data. Here, we present a method to identify the putative parents of polyploids and thus to infer the mode of their origin (auto- versus allopolyploidy) from Amplified Fragment Length Polymorphism (AFLP) data. To this end, we use Cohen's d of distances between in silico polyploids, generated within a priori defined scenarios of origin from a priori delimited putative parental entities (e.g., taxa, genetic lineages), and natural polyploids. Simulations show that the discriminatory power of the proposed method increases mainly with increasing divergence between the lower-ploid putative ancestors and less so with increasing delay of polyploidization relative to the time of divergence. We apply the new method to the Senecio carniolicus aggregate, distributed in the European Alps and comprising two diploid, one tetraploid and one hexaploid species. In the eastern part of its distribution, the S. carniolicus aggregate was inferred to comprise an autopolyploid series, whereas for western populations of the tetraploid species an allopolyploid origin involving the two diploid species was the most likely scenario. Although this suggests that the tetraploid species has two independent origins, other evidence (ribotype distribution, morphology) is consistent with the hypothesis of an autopolyploid origin with subsequent introgression by the second diploid species. Altogether, identifying the best among alternative scenarios using Cohen's d can be straightforward, but particular scenarios, such as allopolyploid origin versus autopolyploid origin with subsequent introgression, remain difficult to be distinguished.

Areas of immediate contact of different cytotypes offer a unique opportunity to study evolutionary dynamics within heteroploid species and to assess isolation mechanisms governing coexistence of cytotypes of different ploidy. The degree of reproductive isolation of cytotypes, i.e., the frequency of heteroploid crosses and subsequent formation of viable and (partly) fertile hybrids, plays a crucial role for the long-term integrity of lineages in contact zones. Here, we assessed fine-scale distribution, spatial clustering and ecological niches as well as patterns of gene-flow in parental and hybrid cytotypes in zones of immediate contact of di-, tetra- and hexaploid Senecio carniolicus (Asteraceae) in the Eastern Alps. Cytotypes were spatially separated also at the investigated micro-scale; the strongest spatial separation was observed for the fully interfertile tetra- and hexaploids. The three main cytotypes showed highly significant niche differences, which were, however, weaker than across their entire distribution ranges in the Eastern Alps. Individuals with intermediate ploidy levels were found neither in the diploid/tetraploid nor in the diploid/hexaploid contact zones indicating strong reproductive barriers. In contrast, pentaploid individuals were frequent in the tetraploid/hexaploid contact zone, albeit limited to a narrow strip in the immediate contact zone of their parental cytotypes. AFLP fingerprinting data revealed introgressive gene flow mediated by pentaploid hybrids from tetra- to hexaploid individuals, but not vice versa. The ecological niche of pentaploids differed significantly from that of tetraploids but not from hexaploids.

Range shifts (especially during the Pleistocene), polyploidisation and hybridization are major factors affecting high-mountain biodiversity. A good system to study their role in the European high mountains is the Doronicum clusii aggregate (Asteraceae), whose four taxa (D. clusii s.s., D. stiriacum, D. glaciale subsp. glaciale and D. glaciale subsp. calcareum) are differentiated geographically, ecologically (basiphilous versus silicicolous) and/or via their ploidy levels (diploid versus tetraploid). Here, we use DNA sequences (three plastid and one nuclear spacer) and AFLP fingerprinting data generated for 58 populations to infer phylogenetic relationships, origin of polyploids—whose ploidy level was confirmed by chromosomally calibrated DNA ploidy level estimates—and phylogeographic history. Taxonomic conclusions were informed, among others, by a Gaussian clustering method for species delimitation using dominant multilocus data. Based on molecular data we identified three lineages: (i) silicicolous diploid D. clusii s.s. in the Alps, (ii) silicicolous tetraploid D. stiriacum in the eastern Alps (outside the range of D. clusii s.s.) and the Carpathians and (iii) the basiphilous diploids D. glaciale subsp. glaciale (eastern Alps) and D. glaciale subsp. calcareum (northeastern Alps); each taxon was identified as distinct by the Gaussian clustering, but the separation of D. glaciale subsp. calcareum and D. glaciale subsp. glaciale was not stable, supporting their taxonomic treatment as subspecies. Carpathian and Alpine populations of D. stiriacum were genetically differentiated suggesting phases of vicariance, probably during the Pleistocene. The origin (autopolyploid versus allopolyploid) of D. stiriacum remained unclear. Doronicum glaciale subsp. calcareum was genetically and morphologically weakly separated from D. glaciale subsp. glaciale but exhibited significantly higher genetic diversity and rarity. This suggests that the more widespread D. glaciale subsp. glaciale originated from D. glaciale subsp. calcareum, which is restricted to a prominent Pleistocene refugium previously identified in other alpine plant species.