Deep into the Aristolochia Flower: Expression of C, D, and E-Class Genes in Aristolochia fimbriata (Aristolochiaceae).

Aristolochia fimbriata (Aristolochiaceae) is a member of an early diverging lineage of flowering plants and a promising candidate for evo-devo studies. Aristolochia flowers exhibit a unique floral synorganization that consists of a monosymmetric and petaloid calyx formed by three congenitally fused sepals, and a gynostemium formed by the congenital fusion between stamens and the stigmatic region of the carpels. This floral ground plan atypical in the magnoliids can be used to evaluate the role of floral organ identity MADS-box genes during early flower evolution. In this study, we present in situ hybridization experiments for the homologs of the canonical C-, D-, and E-class genes. Spatiotemporal expression of the C-class gene AfimAG is restricted to stamens, ovary, and ovules, suggesting a conserved stamen and carpel identity function, consistent with that reported in core-eudicots and monocots. The D-class gene AfimSTK is detected in the anthers, the stigmas, the ovary, the ovules, the fruit, and the seeds, suggesting conserved roles in ovule and seed identity and unique roles in stamens, ovary, and fruit development. In addition, AfimSTK expression patterns in areas of organ abscission and dehiscence zones suggest putative roles linked to senescence processes. We found that both E-class genes are expressed in the anthers and the ovary; however, AfimSEP2 exhibits higher expression compared to AfimSEP1. These findings provide a comprehensive picture of the ancestral expression patterns of the canonical MADS-box floral organ identity genes and the foundations for further comparative analyses in other magnoliids.

Spatio-temporal patterns in pollination of deceptive Aristolochia rotunda L. (Aristolochiaceae).

Pollination success of highly specialised flowers is susceptible to fluctuations of the pollinator fauna. Mediterranean Aristolochia rotunda has deceptive trap flowers exhibiting a highly specialised pollination system. The sole pollinators are kleptoparasitic flies in search of food. This study investigates these pollinators on a spatio-temporal scale and the impact of weather conditions on their availability. Two potential strategies of the plants to cope with pollinator limitation, i.e. autonomous selfing and an increased floral life span, were tested. A total of 6156 flowers were investigated for entrapped pollinators in 10 Croatian populations. Availability of the main pollinator was correlated to meteorological data. Artificial pollination experiments were conducted and the floral life span was recorded in two populations according to pollinator availability. Trachysiphonella ruficeps (Chloropidae) was identified as dominant pollinator, along with less abundant species of Chloropidae, Ceratopogonidae and Milichiidae. Pollinator compositions varied among populations. Weather conditions 15-30 days before pollination had a significant effect on availability of the main pollinator. Flowers were not autonomously selfing, and the floral life span exhibited considerable plasticity depending on pollinator availability. A. rotunda flowers rely on insect pollen vectors. Plants are specialised on a guild of kleptoparasitic flies, rather than on a single species. Pollinator variability may result in differing selection pressures among populations. The availability/abundance of pollinators depends on weather conditions during their larval development. Flowers show a prolonged trapping flower stage that likely increases outcrossing success during periods of pollinator limitation.

Visualization of embolism formation in the xylem of liana stems using neutron radiography.

BACKGROUND AND AIMS: Cold neutron radiography was applied to directly observe embolism in conduits of liana stems with the aim to evaluate the suitability of this method for studying embolism formation and repair. Potential advantages of this method are a principally non-invasive imaging approach with low energy dose compared with synchrotron X-ray radiation, a good spatial and temporal resolution, and the possibility to observe the entire volume of stem portions with a length of several centimetres at one time. METHODS: Complete and cut stems of Adenia lobata, Aristolochia macrophylla and Parthenocissus tricuspidata were radiographed at the neutron imaging facility CONRAD at the Helmholtz-Zentrum Berlin für Materialien und Energie, with each measurement cycle lasting several hours. Low attenuation gas spaces were separated from the high attenuation (water-containing) plant tissue using image processing. KEY RESULTS: Severe cuts into the stem were necessary to induce embolism. The formation and temporal course of an embolism event could then be successfully observed in individual conduits. It was found that complete emptying of a vessel with a diameter of 100 µm required a time interval of 4 min. Furthermore, dehydration of the whole stem section could be monitored via decreasing attenuation of the neutrons. CONCLUSIONS: The results suggest that cold neutron radiography represents a useful tool for studying water relations in plant stems that has the potential to complement other non-invasive methods.

Escaping the lianoid habit: evolution of shrub-like growth forms in Aristolochia subgenus Isotrema (Aristolochiaceae).

PREMISE OF THE STUDY: A large range of growth forms is a notable aspect of angiosperm diversity and arguably a key element of their success. However, few studies within a phylogenetic context have explored how anatomical, developmental, and biomechanical traits are linked with growth form evolution. Aristolochia (∼500 species) consists predominantly of climbers, but a handful of shrub-like species are known from Aristolochia subgenus Isotrema (hereafter, shortened to Isotrema). We test hypotheses proposing that the establishment of functional traits linked to lianescence might limit the ability to evolve structurally diverse growth forms, particularly self-supporting forms. • METHODS: We focus on the origin of the shrub habit in Isotrema, from which we sampled representatives from climbing to self-supporting forms. Morphological, anatomical, and biomechanical characters are optimized on a chloroplast- and nuclear-derived phylogeny. • KEY RESULTS: Character-state reconstructions revealed that the climbing habit is plesiomorphic in Isotrema and shrub-like forms are derived from climbers. However, shrubs do not constitute a monophyletic group. Both shrubs and climbers show large multiseriate rays, but differ in terms of vessel size and proportion of fibers and soft tissues. • CONCLUSION: We suggest that while shrub-like species might have partly escaped from the constraints of life as lianas; their height size and stability are not typical of self-supporting shrubs and trees. Shrubs retained lianoid stem characters that are known to promote flexibility such as ray parenchyma. The transitions to a shrub-like form likely involved relatively simple, developmental changes that may be attributed to heterochronic processes.

Morphological aspects of self-repair of lesions caused by internal growth stresses in stems of Aristolochia macrophylla and Aristolochia ringens.

This study reveals in detail the mechanism of self-repair during secondary growth in the vines Aristolochia macrophylla and Aristolochia ringens based on morphological data. For a comprehensive understanding of the underlying mechanisms during the self-repair of lesions in the sclerenchymatous cylinder of the stem, which are caused by internal growth stresses, a classification of morphological changes in the cells involved in the repair process is required. In an early stage of self-repair, we observed morphological changes as a mere extension of the turgescent cortex cells surrounding the lesion, whereby the cell wall extends locally through visco-elastic/plastic deformation without observable cell wall synthesis. Later stages involve typical cell growth and cell division. Several successive phases of self-repair were investigated by light microscopy of stained samples and confocal laser-scanning microscopy in fluorescence mode. The results indicate that A. macrophylla and A. ringens respond to lesions caused by internal growth stresses with a sophisticated self-repair mechanism comprising several phases of different repair modes.

Structure and biomechanics of trapping flower trichomes and their role in the pollination biology of Aristolochia plants (Aristolochiaceae).

*Catching insects to ensure pollination is one of the most elaborate and specialized mechanisms of insect-plant interactions. Phylogenetically, Aristolochiaceae represent the first angiosperm lineage that developed trap flowers. Here we report the structure and function of specific trichomes contributing to the highly specialized trapping devices. *Investigations were carried out on six Mediterranean Aristolochia species. The morphology and arrangement of the trapping trichomes were investigated by scanning electron microscopy (SEM) and cryo-SEM. To demonstrate frictional anisotropy of the trapping trichome array, a microtribological approach was used. *The results of our experiments support a hypothesis long proposed, but never tested, regarding the trapping mechanism in proterogynous Aristolochia flowers: that an array of highly specialized trichomes arranged eccentrically to the underlying surface is responsible for the easy entrance of insects into flowers but impedes their escape. As they enter the male stage of anthesis, flowers significantly modify their inner surface characteristics, allowing insects to leave. *We have demonstrated the substantial contribution of trapping trichomes to the capture, retention and release of pollinators, an important prerequisite for making cross-pollination possible in most Aristolochia species. Finally, we compare trapping trichomes of Aristolochia with similar structures found in other trapping flowers as well as in pitchers of carnivorous plants not optimized for insect release.

[Specific features of pollination in the Manchurian birthwort Aristolochia manshuriensis].

An analysis of pollination system in Aristolochia manshuriensis has shown that flower structure in this species is strictly adapted to cross-pollination, but the possibility of an autogamous or geitonogamous type of self-pollination with the involvement of insects is not excluded. The flowers of A. manshuriensis are most frequently visited by flies of the family Anthomyiidae, which markedly contribute to their pollination. Males account for 65% of pollinator insects collected from the flowers.

Quantitative and qualitative changes in primary and secondary stem organization of Aristolochia macrophylla during ontogeny: functional growth analysis and experiments.

The anatomy of young and old stems of Aristolochia macrophylla has been investigated for a better understanding of how secondary growth processes cause changes in the stem anatomy of a lianescent plant. In A. macrophylla, following an increase in volume of secondary vascular tissues, the cortical tissues are deformed and the outer sclerenchymatous cylinder ruptures. Morphometric measurements prove that the inner zone of the cortical parenchymatous tissue is compressed prior to the rupture of the outer sclerenchymatous cylinder. After the rupture has occurred, the radial width of the inner primary cortex slightly increases again. This could be caused by strain relaxation, suggesting that the inner primary cortex mechanically behaves similarly to cellular technical foam rubbers. Two different experiments were undertaken to test the outer cortical cylinders mechanically. The outer cortical cylinders comprise the outer sclerenchymatous cortical tissue and a collenchymatous sheath underneath the epidermis and the epidermis. In a first experiment, transverse compression loads were applied to the outside of the cortical cylinders causing ovalization of the cylinder until failure. This experiment allowed the Young's Modulus of the outer cortical cylinders to be determined. In a second set of experiments, radial hydraulic pressure was applied to the inside of the cortical cylinders, mimicking the mechanical effects of internal growth processes. The increase of the internal pressure finally led to rupture of the cortical cylinders. The circumferential stresses acting on the inner surface of the cortical cylinders were calculated. These data allow quantitative estimates of the radial and circumferential pressures effected by vascular secondary growth processes during ontogeny in A. macrophylla stems. The experimental results further indicate that the outer sclerenchymatous cylinder is the main contributor to mechanical stability of young A. macrophylla stems.

Molecular evolution of the petal and stamen identity genes, APETALA3 and PISTILLATA, after petal loss in the Piperales.

Organ loss is an evolutionary phenomenon commonly observed in all kinds of multicellular organisms. Across the angiosperms, petals have been lost several times over the course of their diversification. We examined the evolution of petal and stamen identity genes in the Piperales, a basal lineage of angiosperms that includes the perianthless (with no petals or sepals) families Piperaceae and Saururaceae as well as the Aristolochiaceae, which exhibit a well-developed perianth. Here, we provide evidence for relaxation of selection on the putative petal and stamen identity genes, homologs of APETALA3 and PISTILLATA, following the loss of petals in the Piperales. Our results are particularly interesting as the B-class genes are not only responsible for the production of petals but are also central to stamen identity, the male reproductive organs that show no modification in these plants. Relaxed purifying selection after the loss of only one of these organs suggests that there has been dissociation of the functional roles of these genes in the Piperales.