Jürg Schönenberger

MOLECULAR PHYLOGENETICS AND PATTERNS OF FLORAL EVOLUTION IN THE ERICALES

The diverse and species‐rich order Ericales has found considerable interest among systematists in recent years. Molecular phylogenetic studies not only have convincingly demonstrated the monophyly of the order, comprising 23 families formerly placed in three different subclasses (Asteridae, Dilleniidae, and Rosidae), but have also resolved Ericales as sister to euasterids. Most ericalean families are well circumscribed and have been or are currently subject to intrafamilial phylogenetic studies. In spite of all the attention that Ericales have received recently, there remains a major challenge, the still largely unresolved deeper nodes in the ericalean phylogeny. This study aims to improve our current knowledge of the interfamilial relationships by expanding on gene and taxon sampling and to evaluate the evolution of important floral characters in light of the resulting phylogeny. We add a nuclear region (26s rDNA) to already published data sets (nuclear: 18s rDNA; mitochondrial: atp1, matR; chloroplast: atpB, ndhF, rbcL, matK, the rps16 intron, the trnT‐trnF spacer, and the trnV‐atpE spacer), for a total of 11 molecular markers that include nearly 20 kb of sequences. Our analyses, applying both maximum parsimony and Bayesian inference, resolve some of the deeper nodes in the phylogeny. Strongly supported groups, previously unrecognized or only weakly supported, include (1) a clade comprising all families except Balsaminaceae, Tetrameristaceae, Marcgraviaceae, Fouquieriaceae, Polemoniaceae, and Lecythidaceae; (2) a clade with Sapotaceae, Ebenaceae, and the primuloid families; (3) a clade with Symplocaceae, Styracaceae, and Diapensiaceae; and (4) a clade comprising the latter three families plus Theaceae, Roridulaceae, Actinidiaceae, Sarraceniaceae, Clethraceae, Cyrillaceae, and Ericaceae. At an analytical level, our results indicate that more data in the form of additional markers do improve resolution and branch support and should eventually lead to a fully resolved ericalean phylogeny. At the level of floral evolution, we demonstrate that sympetaly is a homoplasious character in the order, that a diplostemonous floral ground plan likely arose from haplostemonous flowers in Ericales, and that the combination of ovules with a single integument and cellular endosperm formation is characteristic for two of the major clades in the order.

DID CRYPTERONIACEAE REALLY DISPERSE OUT OF INDIA? MOLECULAR DATING EVIDENCE FROM rbcL, ndhF, AND rpl16 INTRON SEQUENCES

Biogeographical and paleontological studies indicated that some ancient Gondwanan taxa have been carried by the rafting Indian plate from Gondwana to Asia. During this journey, the Indian island experienced dramatic latitudinal and climatic changes that caused massive extinctions in its biota. However, some taxa survived these conditions and dispersed “out of India” into South and Southeast Asia, after India collided with the Asian continent in the Early Tertiary. To test this hypothesis, independent estimates for lineage ages are needed. A published rbcL tree supported the sister group relationship between the South and Southeast Asian Crypteroniaceae (comprising Crypteronia, Axinandra, and Dactylocladus) and a clade formed by the African Oliniaceae, Penaeaceae, and Rhynchocalycaceae and the Central and South American Alzateaceae. Molecular dating estimates indicated that Crypteroniaceae split from their West Gondwanan sister clade in the Early to Middle Cretaceous and reached Asia by rafting on the Indian plate. Here we present molecular evidence from additional chloroplast DNA regions and more taxa to test the validity of the out‐of‐India hypothesis for Crypteroniaceae. Both clock‐based (Langley‐Fitch) and clock‐independent age estimates (nonparametric rate smoothing and penalized likelihood) based on maximum likelihood analyses of three chloroplast DNA regions (rbcL, ndhF, and rpl16 intron) were used to infer the age of Crypteroniaceae. Our dating results indicate an ancient Gondwanan origin of Crypteroniaceae in the Early to Middle Cretaceous, followed by diversification on the Indian plate in the Early Tertiary and subsequent dispersal to Southeast Asia. These findings are congruent with recent molecular, paleontological, and biogeographic results in vertebrates. Within the biogeographic context of this study, we explore the critical assignment of paleobotanic and geologic constraints to calibrate ultrametric trees.

Plant Tissues in 3D via X-Ray Tomography: Simple Contrasting Methods Allow High Resolution Imaging

Computed tomography remains strongly underused in plant sciences despite its high potential in delivering detailed 3D phenotypical information because of the low X-ray absorption of most plant tissues. Existing protocols to study soft tissues display poor performance, especially when compared to those used on animals. More efficient protocols to study plant material are therefore needed. Flowers of Arabidopsis thaliana and Marcgravia caudata were immersed in a selection of contrasting agents used to treat samples for transmission electron microscopy. Grayscale values for floral tissues and background were measured as a function of time. Contrast was quantified via a contrast index. The thick buds of Marcgravia were scanned to determine which contrasting agents best penetrate thick tissues. The highest contrast increase with cytoplasm-rich tissues was obtained with phosphotungstate, whereas osmium tetroxide and bismuth tatrate displayed the highest contrast increase with vacuolated tissues. Phosphotungstate also displayed the best sample penetration. Furthermore, infiltration with phosphotungstate allowed imaging of all plants parts at a high resolution of 3 µm, which approaches the maximum resolution of our equipment: 1.5 µm. The high affinity of phosphotungstate for vasculature, cytoplasm-rich tissue, and pollen causes these tissues to absorb more X-rays than the surrounding tissues, which, in turn, makes these tissues appear brighter on the scan data. Tissues with different brightness can then be virtually dissected from each other by selecting the bracket of grayscale to be visualized. Promising directions for the future include in silico phenotyping and developmental studies of plant inner parts (e.g., ovules, vasculature, pollen, and cell nuclei) via virtual dissection as well as correlations of quantitative phenotypes with omics datasets. Therefore, this work represents a crucial improvement of previous methods, allowing new directions of research to be undertaken in areas ranging from morphology to systems biology.

Endressianthus, a New Normapolles‐Producing Plant Genus of Fagalean Affinity from the Late Cretaceous of Portugal

New Normapolles reproductive structures are described from two Late Cretaceous localities, Mira and Esgueira (Olho de Agua), in the Beira Litoral region of western Portugal. They are assigned to a new genus, Endressianthus, containing two new species, Endressianthus miraensis and Endressianthus foveocarpus. The new genus is based on several small inflorescence fragments with cymose branching and dichasial inflorescence subunits bearing either pistillate or staminate flowers, as well as on numerous isolated stamens and fruits. The pistillate flowers have an inferior ovary and minute, acicular tepals. The gynoecium is bicarpellate and syncarpous. In the fruiting stage, the stigmatic area on top of the ovary is seen as an elongate slit that is covered by stiff simple trichomes. Fruits are nuts with one or two locules and one or two seeds. The staminate flowers consist of four stamens with short filaments and elongate tetrasporangiate anthers and apparently a whorl of small, thornlike tepals. Typically, only one to three stamens develop into maturity while the other stamens in the flower abort. Pollen grains in situ show close similarity to dispersed pollen of the genus Interporopollenites. They are oblate with triangular equatorial contour, rounded protruding germinals, three endopores and six exopores, and a distinct arcus that interconnects the germinals. Organization of the inflorescence and flowers as well as the structure of the pollen indicates that this new Normapolles flower is of Fagalean affinity. The newly described taxon is apparently particularly close to members of the Betulaceae and may represent an extinct lineage at the root of the Betulaceae.

The ancestral flower of angiosperms and its early diversification

Recent advances in molecular phylogenetics and a series of important palaeobotanical discoveries have revolutionized our understanding of angiosperm diversification. Yet, the origin and early evolution of their most characteristic feature, the flower, remains poorly understood. In particular, the structure of the ancestral flower of all living angiosperms is still uncertain. Here we report model-based reconstructions for ancestral flowers at the deepest nodes in the phylogeny of angiosperms, using the largest data set of floral traits ever assembled. We reconstruct the ancestral angiosperm flower as bisexual and radially symmetric, with more than two whorls of three separate perianth organs each (undifferentiated tepals), more than two whorls of three separate stamens each, and more than five spirally arranged separate carpels. Although uncertainty remains for some of the characters, our reconstruction allows us to propose a new plausible scenario for the early diversification of flowers, leading to new testable hypotheses for future research on angiosperms.

Structure and Development of the Flowers in Mendoncia, Pseudocalyx, and Thunbergia (Acanthaceae) and Their Systematic Implications

Floral structure and development of several species of the two acanthaceous subfamilies, Mendoncioideae (examined genus Mendoncia) and Thunbergioideae (examined genera Pseudocalyx and Thunbergia), were studied comparatively to evaluate their systematic relationship. Although the fruits are quite different (four-seeded capsules in Thunbergioideae and one- or two-seeded drupes in Mendoncioideae), development shows that there are no differences in the bauplan of the gynoecia. In Mendoncia species, two ovary locules and four ovules are initiated, although at anthesis the adaxial locule and its two ovules are no longer distinguishable The floral development of the different species is also uniform in other respects. Special differentiation of the stamens, such as lignified bristles on anthers, lack of an endothecial cell layer, elongated connective tips, and poricidal opening of the thecae further connect the two subfamilies. Floral structure and development of Mendoncia, Pseudocalyx, and Thunbergia strongly suggest that these genera form a natural group.

Diversity and evolution of floral structure among early diverging lineages in the Ericales

This is a combination of review and original data on floral structure and diversity in the two earliest diverging lineages of the Ericales, i.e. the balsaminoids, comprising Balsaminaceae, Marcgraviaceae and Tetrameristaceae, and the polemonioids, comprising Fouquieriaceae and Polemoniaceae. Each clade is strongly supported in molecular studies, while structural synapomorphies have largely been lacking. For the balsaminoid families, we compare floral morphology, anatomy and histology among selected taxa and find that the entire clade is strongly supported by the shared presence of nectariferous tissue in the floral periphery, thread-like structures on anthers, truncate stigmas, secretion in the ovary, as well as mucilage cells, raphides and tannins in floral tissues. A possible sister group relationship between Balsaminaceae and Tetrameristaceae is supported by the shared presence of post-genital fusion of filaments and ovary and a star-shaped stylar canal. For polemonioids, we document unexpected diversity of floral features in Polemoniaceae, partly providing structural links to Fouquieriaceae. Features include cochlear and quincuncial corolla aestivation, connective protrusions, ventrifixed anthers and nectariferous tissue in the base of the ovary. In addition, we outline future directions for research on floral structure in the Ericales and briefly discuss the general importance of structural studies for our understanding of plant phylogeny and evolution.

Glandulocalyx upatoiensis, a fossil flower of Ericales (Actinidiaceae/Clethraceae) from the Late Cretaceous (Santonian) of Georgia, USA.

BACKGROUND AND AIMS Ericales are a major group of extant asterid angiosperms that are well represented in the Late Cretaceous fossil record, mainly by flowers, fruits and seeds. Exceptionally well preserved fossil flowers, here described as Glandulocalyx upatoiensis gen. & sp. nov., from the Santonian of Georgia, USA, yield new detailed evidence of floral structure in one of these early members of Ericales and provide a secure basis for comparison with extant taxa. METHODS The floral structure of several fossil specimens was studied by scanning electron microscopy (SEM), light microscopy of microtome thin sections and synchrotron-radiation X-ray tomographic microscopy (SRXTM). For direct comparisons with flowers of extant Ericales, selected floral features of Actinidiaceae and Clethraceae were studied with SEM. KEY RESULTS Flowers of G. upatoiensis have five sepals with quincuncial aestivation, five free petals with quincuncial aestivation, 20-28 stamens arranged in a single series, extrorse anther orientation in the bud, ventral anther attachment and a tricarpellate, syncarpous ovary with three free styles and numerous small ovules on axile, protruding-diffuse and pendant placentae. The calyx is characterized by a conspicuous indumentum of large, densely arranged, multicellular and possibly glandular trichomes. CONCLUSIONS Comparison with extant taxa provides clear evidence for a relationship with core Ericales comprised of the extant families Actinidiaceae, Roridulaceae, Sarraceniaceae, Clethraceae, Cyrillaceae and Ericaceae. Within this group, the most marked similarities are with extant Actinidiaceae and, to a lesser degree, with Clethraceae. More detailed analyses of the relationships of Glandulocalyx and other Ericales from the Late Cretaceous will require an improved understanding of the morphological features that diagnose particular extant groups defined on the basis of molecular data.

Floral structure and organization in Platanaceae.

Developing and mature inflorescences and flowers of several representatives of Platanus were studied to clarify various aspects of floral structure and organization. Special attention was given to perianth differentiation. Extant Platanaceae are monoecious with unisexual flowers aggregated into compact, spherical inflorescence heads. Development of hairs in a basipetal direction subdivides the undifferentiated inflorescence surface into floral zones. Development of both male and female flowers of Platanus × hispanica begins with the initiation of a perianth whorl. Thereafter, the reproductive organs emerge on the floral apex: stamens in male flowers, staminodes and carpels in female flowers. The last organs to appear in both sexes are the small organs located between perianth and androecium. At anthesis, in both male and female flowers, organs of the first whorl are inconspicuous, scalelike, and only two to three cell layers thick. Alternating with these first thin organs is a whorl of short but fleshy organs. These second‐whorl organs are basally united with the stamens, forming a short androecial tube. They also show some structural similarities with stamens. These features support the hypothesis that the second‐whorl organs are of androecial (staminodial) origin. This hypothesis is further supported by the fossil record, where, in some taxa, second‐whorl organs are particularly similar to stamens, as well as by morphological comparisons with flowers of Proteaceae.

Genic rather than genome-wide differences between sexually deceptive Ophrys orchids with different pollinators

High pollinator specificity and the potential for simple genetic changes to affect pollinator attraction make sexually deceptive orchids an ideal system for the study of ecological speciation, in which change of flower odour is likely important. This study surveys reproductive barriers and differences in floral phenotypes in a group of four closely related, coflowering sympatric Ophrys species and uses a genotyping‐by‐sequencing (GBS) approach to obtain information on the proportion of the genome that is differentiated between species. Ophrys species were found to effectively lack postpollination barriers, but are strongly isolated by their different pollinators (floral isolation) and, to a smaller extent, by shifts in flowering time (temporal isolation). Although flower morphology and perhaps labellum coloration may contribute to floral isolation, reproductive barriers may largely be due to differences in flower odour chemistry. GBS revealed shared polymorphism throughout the Ophrys genome, with very little population structure between species. Genome scans for FST outliers identified few markers that are highly differentiated between species and repeatable in several populations. These genome scans also revealed highly differentiated polymorphisms in genes with putative involvement in floral odour production, including a previously identified candidate gene thought to be involved in the biosynthesis of pseudo‐pheromones by the orchid flowers. Taken together, these data suggest that ecological speciation associated with different pollinators in sexually deceptive orchids has a genic rather than a genomic basis, placing these species at an early phase of genomic divergence within the ‘speciation continuum’.