Contrasting evolutionary origins of two mountain endemics: Saxifraga wahlenbergii (Western Carpathians) and S. styriaca (Eastern Alps).

BACKGROUND: The Carpathians and the Alps are the largest mountain ranges of the European Alpine System and important centres of endemism. Among the distinctive endemic species of this area is Saxifraga wahlenbergii, a Western Carpathians member of the speciose genus Saxifraga. It was frequently considered a taxonomically isolated Tertiary palaeopolyploid and palaeoendemic, for which the closest relatives could not yet be traced. A recently described narrow endemic of the Eastern Alps, S. styriaca, was hypothesized to be closely related to S. wahlenbergii based on shared presence of peculiar glandular hairs. To elucidate the origin and phylogenetic relationships of both species we studied nuclear and plastid DNA markers based on multiple accessions and analysed the data in a wide taxonomic context. We applied Sanger sequencing, followed by targeted next-generation sequencing (NGS) for a refined analysis of nrITS variants to detect signatures of ancient hybridization. The ITS data were used to estimate divergence times of different lineages using a relaxed molecular clock. RESULTS: We demonstrate divergent evolutionary histories for the two mountain endemics. For S. wahlenbergii we revealed a complicated hybrid origin. Its maternal parent belongs to a Western Eurasian lineage of high mountain taxa grouped in subsect. Androsaceae and is most likely the widespread S. androsacea. The putative second parent was most likely S. adscendens, which belongs to the distantly related subsect. Tridactylites. While Sanger sequencing of nrITS only showed S. adscendens-related variants in S. wahlenbergii, our NGS screening revealed presence of sequences from both lineages with clear predominance of the paternal over the maternal lineage. CONCLUSIONS: Saxifraga styriaca was unambiguously assigned to subsect. Androsaceae and is not the sister taxon of S. wahlenbergii. Accordingly, the similarity of the glandular hairs observed in both taxa rests on parallelism and both species do not constitute an example of a close evolutionary link between the floras of the Western Carpathians and Eastern Alps. With the origin of its paternal, S. adscendens-like ITS DNA estimated to ca. 4.7 Ma, S. wahlenbergii is not a relict of the mid-Tertiary climate optimum. Its hybrid origin is much younger and most likely took place in the Pleistocene.

High mountain origin, phylogenetics, evolution, and niche conservatism of arctic lineages in the hemiparasitic genus Pedicularis (Orobanchaceae).

The origin of the arctic flora covering the northernmost treeless areas is still poorly understood. Arctic plants may have evolved in situ or immigrated from the adjacent ecosystems. Frequently arctic species have disjunctive distributions between the Arctic and high mountain systems of the temperate zone. This pattern may result from long distance dispersal or from glacial plant migrations and extinctions of intermediate populations. The hemiparasitic genus Pedicularis is represented in the Arctic by c. 28 taxa and ranks among the six most species-rich vascular plant genera of this region. In this study, we test the hypothesis that these lineages evolved from predecessors occurring in northern temperate mountain ranges, many of which are current centers of diversity for the genus. We generated a nuclear ribosomal and chloroplast DNA phylogeny including almost all of the arctic taxa and nearly half of the genus as a whole. The arctic taxa of Pedicularis evolved 12-14 times independently and are mostly nested in lineages that otherwise occur in the high mountains of Eurasia and North America. It appears that only three arctic lineages arose from the present-day center of diversity of the genus, in the Hengduan Mountains and Himalayas. Two lineages are probably of lowland origin. Arctic taxa of Pedicularis show considerable niche conservatism with respect to soil moisture and grow predominantly in moist to wet soils. The studied characteristics of ecology, morphology, and chromosome numbers of arctic Pedicularis show a heterogeneous pattern of evolution. The directions of morphological changes among the arctic lineages show opposing trends. Arctic taxa are chiefly diploid, the few tetraploid chromosome numbers of the genus were recorded only for arctic taxa. Five arctic Pedicularis are annuals or biennials, life forms otherwise rare in the Arctic. Other genera of the Orobanchaceae consist also of an elevated number of short-lived species, thus hemiparasitism may favor this life form in the Arctic.

Repeated upslope biome shifts in Saxifraga during late-Cenozoic climate cooling.

Mountains are among the most biodiverse places on Earth, and plant lineages that inhabit them have some of the highest speciation rates ever recorded. Plant diversity within the alpine zone - the elevation above which trees cannot grow-contributes significantly to overall diversity within mountain systems, but the origins of alpine plant diversity are poorly understood. Here, we quantify the processes that generate alpine plant diversity and their changing dynamics through time in Saxifraga (Saxifragaceae), an angiosperm genus that occurs predominantly in mountain systems. We present a time-calibrated molecular phylogenetic tree for the genus that is inferred from 329 low-copy nuclear loci and incorporates 73% (407) of known species. We show that upslope biome shifts into the alpine zone are considerably more prevalent than dispersal of alpine specialists between regions, and that the rate of upslope biome shifts increased markedly in the last 5 Myr, a timeframe concordant with a cooling and fluctuating climate that is likely to have increased the extent of the alpine zone. Furthermore, alpine zone specialists have lower speciation rates than generalists that occur inside and outside the alpine zone, and major speciation rate increases within Saxifraga significantly pre-date increased rates of upslope biome shifts. Specialisation to the alpine zone is not therefore associated with speciation rate increases. Taken together, this study presents a quantified and broad scale perspective of processes underpinning alpine plant diversity.

Untargeted Metabolomics for Integrative Taxonomy: Metabolomics, DNA Marker-Based Sequencing, and Phenotype Bioimaging.

Integrative taxonomy is a fundamental part of biodiversity and combines traditional morphology with additional methods such as DNA sequencing or biochemistry. Here, we aim to establish untargeted metabolomics for use in chemotaxonomy. We used three thallose liverwort species Riccia glauca, R. sorocarpa, and R. warnstorfii (order Marchantiales, Ricciaceae) with Lunularia cruciata (order Marchantiales, Lunulariacea) as an outgroup. Liquid chromatography high-resolution mass-spectrometry (UPLC/ESI-QTOF-MS) with data-dependent acquisition (DDA-MS) were integrated with DNA marker-based sequencing of the trnL-trnF region and high-resolution bioimaging. Our untargeted chemotaxonomy methodology enables us to distinguish taxa based on chemophenetic markers at different levels of complexity: (1) molecules, (2) compound classes, (3) compound superclasses, and (4) molecular descriptors. For the investigated Riccia species, we identified 71 chemophenetic markers at the molecular level, a characteristic composition in 21 compound classes, and 21 molecular descriptors largely indicating electron state, presence of chemical motifs, and hydrogen bonds. Our untargeted approach revealed many chemophenetic markers at different complexity levels that can provide more mechanistic insight into phylogenetic delimitation of species within a clade than genetic-based methods coupled with traditional morphology-based information. However, analytical and bioinformatics analysis methods still need to be better integrated to link the chemophenetic information at multiple scales.

Molecular Phylogenetics and Micromorphology of Australasian Stipeae (Poaceae, Subfamily Pooideae), and the Interrelation of Whole-Genome Duplication and Evolutionary Radiations in This Grass Tribe.

The mainly Australian grass genus Austrostipa (tribe Stipeae) comprising approximately 64 species represents a remarkable example of an evolutionary radiation. To investigate aspects of diversification, macro- and micromorphological variation in this genus, we conducted molecular phylogenetic and scanning electron microscopy (SEM) analyses including representatives from most of Austrostipa's currently accepted subgenera. Because of its taxonomic significance in Stipeae, we studied the lemma epidermal pattern (LEP) in 34 representatives of Austrostipa. Plastid DNA variation within Austrostipa was low and only few lineages were resolved. Nuclear ITS and Acc1 yielded comparable groupings of taxa and resolved subgenera Arbuscula, Petaurista, and Bambusina in a common clade and as monophyletic. In most of the Austrostipa species studied, the LEP was relatively uniform (typical maize-like), but six species had a modified cellular structure. The species representing subgenera Lobatae, Petaurista, Bambusina as well as A. muelleri from subg. Tuberculatae were well-separated from all the other species included in the analysis. We suggest recognizing nine subgenera in Austrostipa (with number of species): Arbuscula (4), Aulax (2), Austrostipa (36), Bambusina (2), Falcatae (10), Lobatae (5), Longiaristatae (2), Petaurista (2) and the new subgenus Paucispiculatae (1) encompassing A. muelleri. Two paralogous sequence copies of Acc1, forming two distinct clades, were found in polyploid Austrostipa and Anemanthele. We found analogous patterns for our samples of Stipa s.str. with their Acc1 clades strongly separated from those of Austrostipa and Anemanthele. This underlines a previous hypothesis of Tzvelev (1977) that most extant Stipeae are of hybrid origin. We also prepared an up-to-date survey and reviewed the chromosome number variation for our molecularly studied taxa and the whole tribe Stipeae. The chromosome base number patterns as well as dysploidy and whole-genome duplication events were interpreted in a phylogenetic framework. The rather coherent picture of chromosome number variation underlines the enormous phylogenetic and evolutionary significance of this frequently ignored character.

Parallel evolutionary patterns in multiple lineages of arctic Artemisia L. (Asteraceae).

Early observers of plant evolution in the Arctic have noted a floristic similarity with temperate alpine regions and a predominance of high ploidy levels. The aim of our study was to survey these and other traits in multiple closely related but independently evolved lineages of Artemisia. Our phylogenetic study was based on 133 taxa using 3'-ETS and ITS, and on data on morphology, karyology, distribution, and ecological preferences. We compared Arctic lineages with sister groups and tested whether patterns were significantly different. We found: (1) Artemisia has independently adapted to Arctic habitats 13-18 times; (2) There were no ecological preferences of putative progenitors that might determine the colonization success in the Arctic, although most sister groups were centered in steppe habitats; (3) Plant height was distinctly reduced in Arctic lineages; (4) Arctic lineages contained no more polyploids than their respective sister groups or taxa from other habitats; (5) Enlarged flower heads have evolved repeatedly, probably for better pollinator attraction. This strategy could be a substitute for polyploidy, which is typical in other Arctic taxa. Stronger pollinator attraction should result in better outcrossing and higher heterozygosity in the offspring, which is among the main effects of polyploidy.

SOURCES OF THE ARCTIC FLORA: ORIGINS OF ARCTIC SPECIES IN RANUNCULUS AND RELATED GENERA.

The arctic biome is a relatively young ecosystem with ~2300 species of vascular plants. We studied the genus Ranunculus as an example of the origin and evolution of the arctic flora. For this purpose we used molecular phylogenetic and clock analyses based on evaluation of nuclear ITS and chloroplast matK-trnK DNA sequences in 194 taxa of Ranunculus and closely related genera. Taxa occurring in the Arctic arose form seven phylogenetic lineages of Ranunculus and also in the genera Coptidium and Halerpestes. Two clades of Ranunculus are species-rich in the Arctic, i.e., Ranunculus sect. Ranunculus and R. sect. Auricomus (both from R. subg. Ranunculus), but this is due to a number of arctic "microtaxa" morphologically barely separate from R. acris in the former clade and the widely agamospermic species complex of R. auricomus in the latter. Lineages with species adapted to wetlands or aquatic habitats are significant groups represented in the arctic flora (R. subg. Ranunculus sectt. Flammula and Hecatonia/Xanthobatrachium, R. subg. Batrachium, genus Coptidium) but show no clear signs of radiation in the Arctic or the northern boreal zone, except for sectt. Hecatonia/Xanthobatrachium, with R. hyperboreus and R. sceleratus subsp. reptabundus. Astonishingly few of the otherwise numerous lineages of Ranunculus with distributions in the higher mountain systems of Eurasia and North America have acted as "founding sources" for the arctic flora. The only clear example is that of the arctic-alpine R. glacialis and the Beringian R. chamissonis from the lineage of subg. R. sectt. Aconitifolii/Crymodes, although there might be others in sect. Auricomus not recovered in the current molecular data. Lineages that gave rise to arctic taxa diverged from each other from the early Miocene (R. glacialis/R. chamissonis, Coptidium, lineages in Halerpestes) and continued at an even rate throughout the Tertiary. There are no signs that the intense climate changes of the late Pliocene and the Quaternary substantially accelerated or impeded diversification in Ranunculus. Only the crown group split of R. acris and its relatives is clearly of Quaternary age. A detailed comparison concerning morphology, karyology, and life form excludes fundamental differences between taxa of Ranunculus in the Arctic and their respective closest relatives in regions south of it. Ecological traits, e.g., preferences for dry or moist soils or growth in open and sheltered conditions, also do not differ between arctic and nonarctic̣ taxa. Migration into the Arctic thus started from different phylogenetic lineages and at different times, without development of obvious special traits in the adaptation to arctic environments. This recurrent pattern in Ranunculus differs from that seen in other arctic genera, e.g., Artemisia, in which special traits of adaptation to arctic environments are found. In Ranunculus, the origin of the open arctic biome primarily favored range expansions of taxa/species already adapted to wet habitats in cold areas and depending on rapid dispersal.

Design, synthesis, computational and biological evaluation of new anxiolytics.

New anxiolytics have been discovered by prediction of biological activity with computer programs pass and derek for a heterogeneous set of 5494 highly chemically diverse heterocyclic compounds (thiazoles, pyrazoles, isatins, a-fused imidazoles and others). The majority of tested compounds exhibit the predicted anxiolytic effect. The most potent activity was found in 2-(4-nitrophenyl)-3-(4-phenylpiperazinomethyl)imidazo[1,2-a]pyridine 8, 1-[(4-bromophenyl)-2-oxoethyl]-3-(1,3-dioxolano)-2-indolinone 3, 5-hydroxy-3-methoxycarbonyl-1-phenylpyrazole 5 and 2-(4-fluorophenyl)-3-(4-methylpiperazinomethyl)imidazo[1,2-a]pyridine 7. The application of the computer-assisted approach significantly reduced the number of synthesized and tested compounds and increased the chance of finding new chemical entities (NCEs).