Relative genome size variation in the African agroforestry tree Parkia biglobosa (Fabaceae: Caesalpinioideae) and its relation to geography, population genetics, and morphology.

Variation in genome size and in chromosome number can be linked to genetic, morphological, and ecological characteristics, and thus be taxonomically significant. We screened the relative genome size (RGS) and counted the number of mitotic chromosomes in the African agroforestry tree Parkia biglobosa, a widely distributed savannah species that shows conspicuous morphological clinal variation and strong genetic structure, and tested for linkage of RGS variation to geography, leaf morphology, and population genetic variation. An improved protocol for the preparation of chromosomes was developed. The study is based on 58 individuals from 15 populations covering most of the distribution range of the species. We observed differences in RGS among individuals of up to 10.2%, with some of the individuals differing statistically in RGS from the bulk of screened individuals. Most of the RGS variation was within populations, whereas variation was unrelated to any of the tested features of the species. Those chromosome numbers that could be exactly established were invariable 2n = 2x = 26. In conclusion, there was no evidence from the karyological data for structured intraspecific taxonomic heterogeneity.

Quality Control of Valerianae Radix by Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy.

(Acetoxy-)valerenic acid and total essential oil content are important quality attributes of pharmacy grade valerian root (Valerianae radix). Traditional analysis of these quantities is time-consuming and necessitates (harmful) solvents. Here we investigated an application of attenuated total reflection Fourier transform infrared spectroscopy for extractionless analysis of these quality attributes on a representative sample comprising 260 wild-crafted individuals covering the Central European taxonomic diversity of the Valeriana officinalis L. s. l. species aggregate with its three major ploidy cytotypes (i.e., di-, tetra- and octoploid). Calibration models were built by orthogonal partial least squares regression for quantitative analysis of (acetoxy-)valerenic acid and total essential oil content. For the latter, we propose a simplistic protocol involving apolar extraction followed by gas chromatography as a reference method for multivariate calibration in order to handle the analysis of samples taken from individual plants. We found good predictive ability of chemometric models for quantification of valerenic acid, acetoxyvalerenic acid, total sesquiterpenoid acid, and essential oil content with a root mean squared error of cross-validation of 0.064, 0.043, and 0.09 and root mean squared error of prediction of 0.066, 0.057, and 0.09 (% content), respectively. Orthogonal partial least squares discriminant analysis revealed good discriminability between the most productive phenotype (i.e., the octoploid cytotype) in terms of sesquiterpenoid acids, and the less productive ones (i.e., di- and tetraploid). All in all, our results demonstrate the application of attenuated total reflection Fourier transform infrared spectroscopy for rapid, extractionless estimation of the most important quality attributes of valerian root and minimally invasive identification of the most productive phenotype in terms of sesquiterpenoid acids.

Pollen precedence in sexual Potentilla puberula and its role as a protective reproductive barrier against apomictic cytotypes.

Cross-pollination is a major factor determining the demographic dynamics of mixed-ploidy populations. Typically, rare cytotypes are suppressed due to reduced female fertility by losing gametes in heteroploid crosses (i.e., through minority cytotype exclusion). In species with reproductive differentiation into sexual and apomictic cytotypes, sexuals might be reproductively suppressed by apomicts (or transformed due to introgression of apomixis genes). Pollen precedence potentially acts as a post-pollination pre-fertilization barrier protecting sexuals against their apomictic counterparts. We estimated the role of pollen precedence as a barrier against cross-fertilization of tetraploid sexuals by penta- and heptaploid gametophytic apomicts in Potentilla puberula (Rosaceae) by means of controlled crosses, and inference of the paternity through DNA ploidy estimation of embryos. Individuals from five regions spanning an elevational and biogeographic gradient were used to account for the variation in the relative frequencies of reproductive modes across the study area. We tested (1) whether the application of heteroploid pollen (sexual × apomictic) causes a reduction of seed yield compared to homoploid crosses (sexual × sexual), and (2) if so, whether pollen precedence recovers the seed yield in simultaneous applications of pollen from sexuals and apomicts (mixed-ploidy). Seed yield was significantly lower in hetero- than in homoploid crosses. We found clear evidence for precedence of homoploid pollen, despite a 13% to 15% of embryos experienced a change in ploidy due to heteroploid fertilizations. Thus, our study indicates that pollen precedence operates as a barrier against intercytotype fertilization in P. puberula, promoting the integrity of the sexual cytotype and their co-existence with apomictic individuals.

Sexual intraspecific recombination but not de novo origin governs the genesis of new apomictic genotypes in Potentilla puberula (Rosaceae).

Apomixis - asexual reproduction via seeds - might arise de novo following polyploidisation events, or via reproductive transfer of apomixis. Both processes can be obtained within species or via hybridisation. We aimed to determine the origin of apomictic genotypes in Potentilla puberula, a rosaceous species showing reproductive differentiation with ploidy: sexual tetraploids and apomictic penta- to octoploids, which regularly co-occur in sympatry. The study is based on 726 individuals, comprising all cytotypes, collected from 138 populations in the Eastern European Alps. We established relationships of cytotypes based on AFLP fingerprinting and cpDNA sequencing to test (1) whether the apomicts are of recurrent allopolyploid origin or originated from within the species via autopolyploidy, and (2) whether there are indications for reproductive transfer versus de novo origin of apomixis. Three principal pathways were identified which explain the origin of new apomictic genotypes, all involving at least one apomictic parent and thus compatible with the idea of reproductive transfer of the apomictic trait to the progeny: (1) self-fertilisation of unreduced egg cells in apomicts; (2) cross-fertilisation among apomicts; and (3) occasionally, heteroploid crosses among sexuals and apomicts. Autopolyploids derived from tetraploid sexuals were repeatedly observed, but did not express apomixis. Finally, our results suggest no role of other species in the origin of extant apomictic genotypes of P. puberula, although local hybrids with P. crantzii were identified. In conclusion, our results show that the formation of new apomictic genotypes required a genetic contribution from at least one apomictic parent. This finding is in accordance with the idea that apomixis is inheritable in P. puberula. On the contrary, lack of apomixis in penta- and hexaploids derived from sexual backgrounds did not support the hypothesis of a de novo origin of apomixis. Relatively high frequency of remnant sexuality in the apomicts involving different cytological pathways of seed formation can explain their high cytological and genotypic diversity. Finally, lack of global introgression from a third taxon is in support of P. puberula as a concise, although highly diverse, species.

Prospects and limits of the flow cytometric seed screen--insights from Potentilla sensu lato (Potentilleae, Rosaceae).

The flow cytometric seed screen allows for identification of reproductive modes of seed formation and inference of the ploidy of contributing gametes. However, the lack of a mathematical formalization to infer male/female genomic contributions, and the prerequisite of a binucleate female contribution to the endosperm limits its applicability. We evaluated this assumption combining a DNA-based progeny survey with a comparison of the cytology of reproductive pathways co-occurring within single individuals representing 14 Potentilleae species from six phylogenetic lineages. A numerical framework valid for sexual and pseudogamous taxa was developed, enabling quantification of female and male genomes contributing to embryo and endosperm independent of gametophyte origins, numbers of sperm involved and ploidy of parents. The inference strongly depended on accurate peak index estimation. The endosperm of Potentilleae species received a binucleate female contribution in five evolutionary lineages whereas endosperm formation remained uncertain in the Tormentillae. A modified flow cytometric seed screen protocol was developed to cope with low endosperm contents. Evolutionary conservation of a binucleate female contribution to the endosperm suggested wide applicability of flow cytometric seed screen - at least in the Potentilleae. However, alternative progeny surveys and precise embryo/endosperm ploidy estimates are required for a comprehensive understanding of the cytology of seed formation.

Implications of hybridisation and cytotypic differentiation in speciation assessed by AFLP and plastid haplotypes--a case study of Potentilla alpicola La Soie.

BACKGROUND: Hybridisation is presumed to be an important mechanism in plant speciation and a creative evolutionary force often accompanied by polyploidisation and in some cases by apomixis. The Potentilla collina group constitutes a particularly suitable model system to study these phenomena as it is morphologically extensively variable, exclusively polyploid and expresses apomixis. In the present study, the alpine taxon Potentilla alpicola has been chosen in order to study its presumed hybrid origin, identify underlying evolutionary processes and infer the discreteness or taxonomic value of hybrid forms. RESULTS: Combined analysis of AFLP, cpDNA sequences and ploidy level variation revealed a hybrid origin of the P. alpicola populations from South Tyrol (Italy) resulting from crosses between P. pusilla and two cytotypes of P. argentea. Hybrids were locally sympatric with at least one of the parental forms. Three lineages of different evolutionary origin comprising two ploidy levels were identified within P. alpicola. The lineages differed in parentage and the complexity of the evolutionary process. A geographically wide-spread lineage thus contrasted with locally distributed lineages of different origins. Populations of P. collina studied in addition, have been regarded rather as recent derivatives of the hexaploid P. argentea. The observation of clones within both P. alpicola and P. collina suggested a possible apomictic mode of reproduction. CONCLUSIONS: Different hybridisation scenarios taking place on geographically small scales resulted in viable progeny presumably stabilised by apomixis. The case study of P. alpicola supports that these processes played a significant role in the creation of polymorphism in the genus Potentilla. However, multiple origin of hybrids and backcrossing are considered to produce a variety of evolutionary spontaneous forms existing aside of reproductively stabilised, established lineages.

Apomixis is not prevalent in subnival to nival plants of the European Alps.

BACKGROUND AND AIMS: High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic species occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown. METHODS: The mode of reproduction in 14 species belonging to seven families was investigated via flow cytometric seed screen. The sampling comprised 12 species typical for nival to subnival plant communities of the European Alps without any previous information on apomixis (Achillea atrata, Androsace alpina, Arabis caerulea, Erigeron uniflorus, Gnaphalium hoppeanum, Leucanthemopsis alpina, Oxyria digyna, Potentilla frigida, Ranunculus alpestris, R. glacialis, R. pygmaeus and Saxifraga bryoides), and two high-alpine species with apomixis reported from other geographical areas (Leontopodium alpinum and Potentilla crantzii). KEY RESULTS: Flow cytometric data were clearly interpretable for all 46 population samples, confirming the utility of the method for broad screenings on non-model organisms. Formation of endosperm in all species of Asteraceae was documented. Ratios of endosperm : embryo showed pseudogamous apomixis for Potentilla crantzii (ratio approx. 3), but sexual reproduction for all other species (ratios approx. 1·5). CONCLUSIONS: The occurrence of apomixis is not correlated to high altitudes, and cannot be readily explained by selective forces due to environmental conditions. The investigated species have probably other adaptations to high altitudes to maintain reproductive assurance via sexuality. We hypothesize that shifts to apomixis are rather connected to frequencies of polyploidization than to ecological conditions.

Relative performance of customized and universal probe sets in target enrichment: A case study in subtribe Malinae.

PREMISE: Custom probe design for target enrichment in phylogenetics is tedious and often hinders broader phylogenetic synthesis. The universal angiosperm probe set Angiosperms353 may be the solution. Here, we test the relative performance of Angiosperms353 on the Rosaceae subtribe Malinae in comparison with custom probes that we specifically designed for this clade. We then address the impact of bioinformatically altering the performance of Angiosperms353 by replacing the original probe sequences with orthologs extracted from the Malus domestica genome. METHODS: To evaluate the relative performance of these probe sets, we compared the enrichment efficiency, locus recovery, alignment length, proportion of parsimony-informative sites, proportion of potential paralogs, the topology and support of the resulting species trees, and the gene tree discordance. RESULTS: Locus recovery was highest for our custom Malinae probe set, and replacing the original Angiosperms353 sequences with a Malus representative improved the locus recovery relative to Angiosperms353. The proportion of parsimony-informative sites was similar between all probe sets, while the gene tree discordance was lower in the case of the custom probes. DISCUSSION: A custom probe set benefits from data completeness and can be tailored toward the specificities of the project of choice; however, Angiosperms353 was equally as phylogenetically informative as the custom probes. We therefore recommend using both a custom probe set and Angiosperms353 to facilitate large-scale systematic studies, where financially possible.

Evidence for Glacial Refugia of the Forest Understorey Species Helleborus niger (Ranunculaceae) in the Southern as Well as in the Northern Limestone Alps.

Glacial refugia of alpine and subnival biota have been intensively studied in the European Alps but the fate of forests and their understory species in that area remains largely unclear. In order to fill this gap, we aimed at disentangling the spatiotemporal diversification of disjunctly distributed black hellebore Helleborus niger (Ranunculaceae). We applied a set of phylogeographic analyses based on restriction-site associated DNA sequencing (RADseq) data and plastid DNA sequences to a range-wide sampling of populations. These analyses were supplemented with species distribution models generated for the present and the Last Glacial Maximum (LGM). We used exploratory analyses to delimit genomically coherent groups and then employed demographic modeling to reconstruct the history of these groups. We uncovered a deep split between two major genetic groups with western and eastern distribution within the Southern Limestone Alps, likely reflecting divergent evolution since the mid-Pleistocene in two glacial refugia situated along the unglaciated southern margin of the Alps. Long-term presence in the Southern Limestone Alps is also supported by high numbers of private alleles, elevated levels of nucleotide diversity and the species' modeled distribution at the LGM. The deep genetic divergence, however, is not reflected in leaf shape variation, suggesting that the morphological discrimination of genetically divergent entities within H. niger is questionable. At a shallower level, populations from the Northern Limestone Alps are differentiated from those in the Southern Limestone Alps in both RADseq and plastid DNA data sets, reflecting the North-South disjunction within the Eastern Alps. The underlying split was dated to ca. 0.1 mya, which is well before the LGM. In the same line, explicit tests of demographic models consistently rejected the hypothesis that the partial distribution area in the Northern Limestone Alps is the result of postglacial colonization. Taken together, our results strongly support that forest understory species such as H. niger have survived the LGM in refugia situated along the southern, but also along the northern or northeastern periphery of the Alps. Being a slow migrator, the species has likely survived repeated glacial-interglacial circles in distributional stasis while the composition of the tree canopy changed in the meanwhile.

A comprehensive chloroplast DNA-based phylogeny of the genus Potentilla (Rosaceae): implications for its geographic origin, phylogeography and generic circumscription.

A reconstruction of phylogenetic relationships based on three chloroplast DNA markers comprising 98 species of the genus Potentilla and 15 additional genera from the tribe Potentilleae (Rosaceae) is presented. The phylogeny supported the current generic concept of the subtribe Fragariinae and resolved major lineages within the subtribe Potentillinae. Resolved lineages corresponded to the combined genera Argentina, Piletophyllum, and Tylosperma, an European and Asian group of species approximately equivalent to the Trichocarpae Herbaceae sensu Th. Wolf, the series Tormentillae Th. Wolf, a North American clade comprising the genera Horkelia, Horkeliella, and Ivesia, the species Potentilla fragarioides and P. freyniana, and to a taxonomically highly diverse but molecularly little diverged core group of Potentilla, respectively. Age estimates of phylogenetic splits resolved in the Potentilleae using Bayesian inference, suggested a diversification of the tribe in the Eocene and radiation of two major evolutionary lineages corresponding to the Fragariinae and Potentillinae at approximately comparable times. Ancestral area reconstructions based on the recent distribution ranges of species and collection sites of cpDNA haplotypes suggested an Asian origin for Potentilla s.str., and explained the arrival of this still informal taxon in Europe and particularly North America by multiple dispersal events. In combining the phylogenetic and geographic data with molecularly inferred time estimates and taxonomy, strongly contrasting evolutionary patterns were identified. These evolutionary patterns included rapid speciation on a continental and worldwide scale accompanied by multiple intercontinental dispersals opposed to largely diverged lineages of limited taxonomic diversity and vicariant geographic distribution. The molecular-based phylogeographic hypothesis finally is discussed on the background of the fossil record of Potentilla.

Occurrence of apomictic conspecifics and ecological preferences rather than colonization history govern the geographic distribution of sexual Potentilla puberula.

The geographic distribution of sexual-apomictic taxa (i.e., comprising individuals usually reproducing either sexually or asexually via seeds) is traditionally thought to be driven by their ecological preferences and colonization histories. Where sexuals and apomicts get into contact with each other, competitive and reproductive interactions can interfere with these factors, an aspect which hitherto received little attention in biogeographic studies. We disentangled and quantified the relative effects of the three factors on the distribution of tetraploid sexuals in Potentilla puberula in a latitudinal transect through the Eastern European Alps, in which they are codistributed with penta-, hepta-, and octoploid apomictic conspecifics. Effects were explored by means of binomial generalized linear regression models combining a single with a multiple predictor approach. Postglacial colonization history was inferred from population genetic variation (AFLPs and cpDNA) and quantified using a cost distance metric. The study was based on 235 populations, which were purely sexual, purely apomictic, or of mixed reproductive mode. The occurrence of apomicts explained most of the variation in the distribution of sexuals (31%). Specifically, the presence of sexual tetraploids was negatively related to the presence of each of the three apomictic cytotypes. Effects of ecological preferences were substantial too (7% and 12% of the total variation explained by ecological preferences alone, or jointly with apomicts' occurrence, respectively). In contrast, colonization history had negligible effects on the occurrence of sexuals. Taken together, our results highlight the potentially high impact of reproductive interactions on the geographic distribution of sexual and apomictic conspecifics and that resultant mutual exclusion interrelates to ecological differentiation, a situation potentially promoting their local coexistence.

Phylogeographic structure of the chloroplast DNA gene pool in North American Boechera--a genus and continental-wide perspective.

Continental-wide phylogeographic studies of plants in North America are rare. In our study we examined the phylogeographic history of Boechera (Brassicaceae) on a continental-wide scale testing if it is possible to do an analysis for 57 of the currently accepted taxa simultaneously. A large amount of haplotype sharing is explained both by recurrent hybridization and by non-differentiation of haplotypes since speciation. Hence, the chloroplast gene pool in Boechera predates speciation and therefore justifies the simultaneous analysis of a large number of taxa. Unrelated from taxon identity we can show that the evolutionary lineages detected have a different phylogeographic history in terms of glacial refugia and recently recolonised areas.

The morphometrics of autopolyploidy: insignificant differentiation among sexual-apomictic cytotypes.

Polyploidization of the plant genome affects the phenotype of individuals including their morphology, i.e. size and form. In autopolyploids, we expect mainly nucleotypic effects, from a number of monoploid genomes (i.e. chromosome sets) or genome size, seen from an increase in size or dimension of the polyploids compared with the diploids (or lower ploids). To identify nucleotypic effects, confounding effects of hybridity (observed in allopolyploids), postpolyploidization processes or environmental effects need to be considered. We morphometrically analysed five ploidy cytotypes of the sexual-apomictic species Potentilla puberula cultivated ex situ under the same experimental conditions. Sexuals are mainly tetraploid, while higher ploidy (penta- to octoploidy) is typically associated with the expression of apomixis. The cytotypes likely arose via autopolyploidization although historic involvement of another species in the origin of apomicts cannot be fully ruled out, suggested by a slight molecular differentiation among reproductive modes. We (i) revisited molecular differentiation using amplified fragment length polymorphisms and performed a morphometric analysis to test (ii) if cytotypes are morphologically differentiated from each other and (iii) if the size of individuals is related to their ploidy. Weak molecular differentiation of sexual versus apomictic individuals was confirmed. Cytotypes and reproductive modes were also morphologically poorly differentiated from each other, i.e. apomicts largely resampled the variation of the sexuals and did not exhibit a unique morphology. Overall size of individuals increased moderately but significantly with ploidy (ca. 14 % in the comparison of octo- with tetraploids). The results support an autopolyploid origin of the P. puberula apomicts and suggest a nucleotypic effect on overall plant size. We discuss taxonomic consequences of the results in the context of data on reproductive relationships among cytotypes and their ecological preferences and evolutionary origin, and conclude that cytotypes are best treated as intraspecific variants within a single species.

Difference in reproductive mode rather than ploidy explains niche differentiation in sympatric sexual and apomictic populations of Potentilla puberula.

Apomicts tend to have larger geographical distributional ranges and to occur in ecologically more extreme environments than their sexual progenitors. However, the expression of apomixis is typically linked to polyploidy. Thus, it is a priori not clear whether intrinsic effects related to the change in the reproductive mode or rather in the ploidy drive ecological differentiation. We used sympatric sexual and apomictic populations of Potentilla puberula to test for ecological differentiation. To distinguish the effects of reproductive mode and ploidy on the ecology of cytotypes, we compared the niches (a) of sexuals (tetraploids) and autopolyploid apomicts (penta-, hepta-, and octoploids) and (b) of the three apomictic cytotypes. We based comparisons on a ploidy screen of 238 populations along a latitudinal transect through the Eastern European Alps and associated bioclimatic, and soil and topographic data. Sexual tetraploids preferred primary habitats at drier, steeper, more south-oriented slopes, while apomicts mostly occurred in human-made habitats with higher water availability. Contrariwise, we found no or only marginal ecological differentiation among the apomictic higher ploids. Based on the pronounced ecological differences found between sexuals and apomicts, in addition to the lack of niche differentiation among cytotypes of the same reproductive mode, we conclude that reproductive mode rather than ploidy is the main driver of the observed differences. Moreover, we compared our system with others from the literature, to stress the importance of identifying alternative confounding effects (such as hybrid origin). Finally, we underline the relevance of studying ecological parthenogenesis in sympatry, to minimize the effects of differential migration abilities.

Phylogeography of a living fossil: pleistocene glaciations forced Ginkgo biloba L. (Ginkgoaceae) into two refuge areas in China with limited subsequent postglacial expansion.

Ginkgo biloba is one of the most famous and therefore intensively studied living fossils. The Ginkgoaceae was suggested to have originated in the early Permian approximately 280mya, and at the height of their worldwide radiation consisted of least 16 genera that were distributed in temperate forests. However, China's G. biloba is the only species of this genus which has survived until today. Using a phylogeographical approach we analyzed the history of this remnant from the past. G. biloba is native to China and has been distributed less than 300 years ago by humans throughout all temperate regions of the world. Separate from this human influence, native Chinese populations of G. biloba have been affected by the cycles of glaciation and deglaciation of the Pleistocene era, similar to many other global plant species. We used molecular markers from the nuclear (AFLPs) and plastid genomes (trnK intron, trnS-trnG intergenic spacer), and based on these data, glacial survival of Gingko populations within two refugia in Southwestern and Eastern China, respectively, is suggested. Specifically, for the first time, this study provides evidence of the existence of a refuge area in Eastern China on the West Tianmu Mountains. We can not support any extensive postglacial range expansion from these refuge areas. Furthermore, AFLP data indicate substantial genetic differentiation among populations from Southwestern, Eastern and Central China. We also hypothesize that this evidence supports that as a long-lived dioecious plant, G. biloba displays high levels of intra-populational genetic diversity that have been maintained in most populations, regardless if they are native or reforested stands. Furthermore, we provide evidence that planted trees from Europe, Japan, Korea and America were introduced multiple times from Eastern China.

Asymmetric reproductive interference: The consequences of cross-pollination on reproductive success in sexual-apomictic populations of Potentilla puberula (Rosaceae).

Apomixis evolves from a sexual background and usually is linked to polyploidization. Pseudogamous gametophytic apomicts, which require a fertilization to initiate seed development, of various ploidy levels frequently co-occur with their lower-ploid sexual ancestors, but the stability of such mixed populations is affected by reproductive interferences mediated by cross-pollination. Thereby, reproductive success of crosses depends on the difference in ploidy levels of mating partners, that is, on tolerance of deviation from the balanced ratio of maternal versus paternal genomes. Quality of pollen can further affect reproductive success in intercytotype pollinations. Cross-fertilization, however, can be avoided by selfing which may be induced upon pollination with mixtures of self- and cross-pollen (i.e., mentor effects). We tested for reproductive compatibility of naturally co-occurring tetraploid sexuals and penta- to octoploid apomicts in the rosaceous species Potentilla puberula by means of controlled crosses. We estimated the role of selfing as a crossing barrier and effects of self- and cross-pollen quality as well as maternal: paternal genomic ratios in the endosperm on reproductive success. Cross-fertilization of sexuals by apomicts was not blocked by selfing, and seed set was reduced in hetero- compared to homoploid crosses. Thereby, seed set was negatively related to deviations from balanced parental genomic ratios in the endosperm. In contrast, seed set in the apomictic cytotypes was not reduced in hetero- compared to homoploid crosses. Thus, apomictic cytotypes either avoided intercytotype cross-fertilization through selfing, tolerated intercytotype cross-fertilizations without negative effects on reproductive success, or even benefitted from higher pollen quality in intercytotype pollinations. Our experiment provides evidence for asymmetric reproductive interference, in favor of the apomicts, with significantly reduced seed set of sexuals in cytologically mixed populations, whereas seed set in apomicts was not affected. Incompleteness of crossing barriers further indicated at least partial losses of a parental genomic endosperm balance requirement.

Suitability of Root and Rhizome Anatomy for Taxonomic Classification and Reconstruction of Phylogenetic Relationships in the Tribes Cardueae and Cichorieae (Asteraceae).

The value of root and rhizome anatomy for the taxonomic characterisation of 59 species classified into 34 genera and 12 subtribes from the Asteraceae tribes Cardueae and Cichorieae was assessed. In addition, the evolutionary history of anatomical characters was reconstructed using a nuclear ribosomal DNA sequence-based phylogeny of the Cichorieae. Taxa were selected with a focus on pharmaceutically relevant species. A binary decision tree was constructed and discriminant function analyses were performed to extract taxonomically relevant anatomical characters and to infer the separability of infratribal taxa, respectively. The binary decision tree distinguished 33 species and two subspecies, but only five of the genera (sampled for at least two species) by a unique combination of hierarchically arranged characters. Accessions were discriminated-except for one sample worthy of discussion-according to their subtribal affiliation in the discriminant function analyses (DFA). However, constantly expressed subtribe-specific characters were almost missing and even in combination, did not discriminate the subtribes. Most anatomical characters showed at least some degree of homoplasious evolution limiting their suitability as phylogenetically informative characters.

Ethnobotanical survey of traditionally used plants in human therapy of east, north and north-east Bosnia and Herzegovina.

AIM OF THE STUDY: The study aims to provide a systematical revision of the traditional use of wild and cultivated plants in north-eastern Bosnia and Herzegovina (Western Balkan Peninsula; Southeast Europe). Thereby, it will extend and complement a recent previous study carried out in middle, southern and western Bosnia and Herzegovina. MATERIAL AND METHODS: Information was collected by performing so called open ethnobotanical interviews. The following data were recorded and systematically assembled in a database: name, age and occupation of the interviewed person; the geographic locality and date of the interview; the name of the used plant; plant parts used; prescription background and preparation procedure as well as indication. Plants mentioned to be used by the informants were collected during field trips done together with the informants and taxonomically determined. The corresponding material was finally deposited in the herbarium of the Department of Pharmacognosy of the University of Vienna for the purpose of documentation. RESULTS: In total, 45 places including villages and mountain areas were visited and 84 persons questioned. 254 wild and cultivated species and 1655 different preparations for the use in traditional human therapy were recorded. The most frequently mentioned indications were disorders of the gastrointestinal tract, respiratory system, urogenital tract, skin, blood system, cardiovascular system, nervous system as well as rheumatism. Infusions were the most frequently prepared formulation. Other applied preparations mentioned with decreasing frequency were decocts, sirups, tinctures, collars, direct application of plants without prior preparation, ointments, freshly pressed juices, oils, powders, fluid unctions, macerations and finally suppositories. Special preparations, typical only for the area of Bosnia and Herzegovina were "mehlems" and some kind of sirup called "dulbe secer" (eng. dulbe sugar). While "mehlems" were already recognized and accordingly discussed for the central, southern and western parts of Bosnia and Herzegovina, "dulbe secer" seems to be known in north-eastern region only. The main compounds of this preparation are sugar or honey, lemon and flowers of one particular species of Rosa (with very small flowers), in Bosnia called "dulbe" rose or "sugar" rose. Prescriptions were verbally delivered for up to more than six generations, traditionally from mother to daughter. For the objective of further analyses and comparisons, the recorded data were inserted in the "VOLKSMED" data base of Austrian prescriptions. CONCLUSIONS: The study showed that there exist considerable similarities in medicinal plant use including indications and type of preparations between the different regions and ethnicities of Bosnia and Herzegovina. Interestingly, there were also only little differences in frequencies of medicinal plant use, indications and preparations between middle, western and southern part Bosnia and Herzegovina on one hand and the eastern, northern and north-eastern part of this county on the other hand. The results also demonstrated the high importance of medicinal plants for the physical health of Bosnian people. 70 of the species reported by Bosnian people were also used in official pharmacy. In addition, a variety of less known plants has been used since ages in traditional therapy of this country and hence may be potential sources for new therapies. Therefore, further pharmaceutical research into this particular and scientifically still underexplored proportion of Bosnian plant biodiversity appears promising and is recommended by the authors.

Ethnobotanical study on medicinal use of wild and cultivated plants in middle, south and west Bosnia and Herzegovina.

AIM OF THE STUDY: The results of an ethnobotanical study on the use of wild and cultivated plants in middle, west and south Bosnia and Herzegovina (Western Balkan Peninsula; Southeast Europe) carried out in the years 2006-2009 are presented. Despite the country's extraordinary high plant diversity with about 3600 known species of vascular plants, plant usage in traditional medicine remained largely unexplored in the past. The purpose of this study was the systematic collection of information about usage of wild and cultivated plants in human therapy from Bosnia and Herzegovina. MATERIALS AND METHODS: Information was gathered by performing so called open ethnobotanical interviews by which data on the name, age and occupation of the interviewed person; the geographic locality and date of the interview; the name of the used plant; part of the plant used; the prescription background and preparation procedure as well as the indication was systematically collected. Plants mentioned to be used by the informants were collected, taxonomically determined and corresponding material was deposited in the herbarium of the Department of Pharmacognosy of the University of Vienna. RESULTS: In total, 34 places including villages and mountain areas were visited and 51 people, known as 'traditional healers' by the rest of the inhabitants, questioned. 228 wild and cultivated species and 730 different preparations for the use in human therapy were recorded. Species of the genera Achillea, Hypericum, Mentha, Teucrium, Thymus, and Urtica were particularly highly recommended by the majority of the informants. The most frequently mentioned indications were urogenital tract disorders, respiratory system disorders, gastrointestinal tract disorders, skin ailments, blood system disorders, nervous system disorders, cardiovascular system disorders, and rheumatism. Infusions were the most frequently prepared formulation. Other applied preparations mentioned with decreasing frequency were decocts, ointments, direct application of plants without prior preparation, fluid unctions, sirups and tinctures or collars, freshly pressed juices, powders, and finally macerations. Balms known as "mehlems" were special to Bosnia and were prepared from freshly chopped or freshly pressed herbal parts of various plants. Warmed resins from Abies or Picea species, bees wax, raw cow or pig lard, olive oil and honey were used as additives in the mehlem formulations. Representatives of the genera Arctium, Carlina, Euphrasia, Hypericum, Plantago, Teucrium, and Urtica were most frequently used in these balms. Prescriptions were verbally delivered, usually from mother to daughter and for even more than six generations. For the purpose of further analyses and comparisons, the collected data were inserted in the so called "VOLKSMED" data base of Austrian prescriptions. CONCLUSIONS: The identified broad variety of indications and their frequent applications suggests that traditional plant use is of high importance and still crucial for the medicinal accommodation of Bosnian people. Fifty-seven of the species reported by Bosnian people were used in official pharmacy. In addition, a variety of less known plants has been used since ages in traditional therapy of this country and hence may be potential sources for new therapies. Therefore, further pharmaceutical research into this particular and scientifically still underexplored proportion of Bosnian plant biodiversity appears promising and is recommended by the authors.

Supernetwork identifies multiple events of plastid trnF(GAA) pseudogene evolution in the Brassicaceae.

The occurrence of nonfunctional trnF pseudogenes has been rarely described in flowering plants. However, we describe the first large-scale supernetwork for the Brassiccaeae built from gene trees for 5 loci (adh, chs, matK, trnL-F, and ITS) and report multiple independent origins for trnF pseudogenes in crucifers. The duplicated regions of the original trnF gene are comprised of its anticodon domain and several other highly structured motifs not related to the original gene. Length variation of the trnL-F intergenic spacer region in different taxa ranges from 219 to 900 bp as a result of differences in pseudocopy number (1-14). It is speculated that functional constraints favor 2-3 or 5-6 copies, as found in Arabidopsis and Boechera. The phylogenetic distribution of microstructural changes for the trnL-F region supports ancient patterns of divergence in crucifer evolution for some but not all gene loci.