Evolution of pollination syndromes and corolla symmetry in Balsaminaceae reconstructed using phylogenetic comparative analyses.

BACKGROUND AND AIMS: Floral diversity as a result of plant-pollinator interactions can evolve by two distinct processes: shifts between pollination systems or divergent use of the same pollinator. Although both are pollinator driven, the mode, relative importance and interdependence of these different processes are rarely studied simultaneously. Here we apply a phylogenetic approach using the Balsaminaceae (including the species-rich genus Impatiens) to simultaneously quantify shifts in pollination syndromes (as inferred from the shape and colour of the perianth), as well as divergent use of the same pollinator (inferred from corolla symmetry). METHODS: For 282 species we coded pollination syndromes based on associations between floral traits and known pollination systems, and assessed corolla symmetry. The evolution of these traits was reconstructed using parsimony- and model-based approaches, using phylogenetic trees derived from phylogenetic analyses of nuclear ribosomal and plastid DNA sequence data. KEY RESULTS: A total of 71 % of studied species have a bee pollination syndrome, 22 % a bimodal syndrome (Lepidoptera and bees), 3 % a bird pollination syndrome and 5 % a syndrome of autogamy, while 19 % of species have an asymmetrical corolla. Although floral symmetry and pollination syndromes are both evolutionarily labile, the latter shifts more frequently. Shifts in floral symmetry occurred mainly in the direction towards asymmetry, but there was considerable uncertainty in the pattern of shift direction for pollination syndrome. Shifts towards asymmetrical flowers were associated with a bee pollination syndrome. CONCLUSION: Floral evolution in Impatiens has occurred through both pollination syndrome shifts and divergent use of the same pollinator. Although the former appears more frequent, the latter is likely to be underestimated. Shifts in floral symmetry and pollination syndromes depend on each other but also partly on the region in which these shifts take place, suggesting that the occurrence of pollinator-driven evolution may be determined by the availability of pollinator species at large geographical scales.

Exploring the evolutionary origin of floral organs of Erycina pusilla, an emerging orchid model system.

BACKGROUND: Thousands of flowering plant species attract pollinators without offering rewards, but the evolution of this deceit is poorly understood. Rewardless flowers of the orchid Erycina pusilla have an enlarged median sepal and incised median petal ('lip') to attract oil-collecting bees. These bees also forage on similar looking but rewarding Malpighiaceae flowers that have five unequally sized petals and gland-carrying sepals. The lip of E. pusilla has a 'callus' that, together with winged 'stelidia', mimics these glands. Different hypotheses exist about the evolutionary origin of the median sepal, callus and stelidia of orchid flowers. RESULTS: The evolutionary origin of these organs was investigated using a combination of morphological, molecular and phylogenetic techniques to a developmental series of floral buds of E. pusilla. The vascular bundle of the median sepal indicates it is a first whorl organ but its convex epidermal cells reflect convergence of petaloid features. Expression of AGL6 EpMADS4 and APETALA3 EpMADS14 is low in the median sepal, possibly correlating with its petaloid appearance. A vascular bundle indicating second whorl derivation leads to the lip. AGL6 EpMADS5 and APETALA3 EpMADS13 are most highly expressed in lip and callus, consistent with current models for lip identity. Six vascular bundles, indicating a stamen-derived origin, lead to the callus, stelidia and stamen. AGAMOUS is not expressed in the callus, consistent with its sterilization. Out of three copies of AGAMOUS and four copies of SEPALLATA, EpMADS22 and EpMADS6 are most highly expressed in the stamen. Another copy of AGAMOUS, EpMADS20, and the single copy of SEEDSTICK, EpMADS23, are most highly expressed in the stelidia, suggesting EpMADS22 may be required for fertile stamens. CONCLUSIONS: The median sepal, callus and stelidia of E. pusilla appear to be derived from a sepal, a stamen that gained petal identity, and stamens, respectively. Duplications, diversifying selection and changes in spatial expression of different MADS-box genes shaped these organs, enabling the rewardless flowers of E. pusilla to mimic an unrelated rewarding flower for pollinator attraction. These genetic changes are not incorporated in current models and urge for a rethinking of the evolution of deceptive flowers.

The relationship between nectaries and floral architecture: a case study in Geraniaceae and Hypseocharitaceae.

Background and Aims: Flowers of Geraniaceae and Hypseocharitaceae are generally considered as morphologically simple. However, previous studies indicated complex diversity in floral architecture including tendencies towards synorganization. Most of the species have nectar-rewarding flowers which makes the nectaries a key component of floral organization and architecture. Here, the development of the floral nectaries is studied and placed into the context of floral architecture. Methods: Seven species from Geraniaceae and one from Hypseocharitaceae were investigated using scanning electron microscopy and light microscopy. Samples were prepared and processed using standard protocols. Key Results: The development of the nectary glands follows the same trajectory in all species studied. Minor differences occur in the onset of nectarostomata development. The most striking finding is the discovery that a short anthophore develops via intercalary growth at the level of the nectary glands. This anthophore lifts up the entire flower apart from the nectary gland itself and thus plays an important role in floral architecture, especially in the flowers of Pelargonium. Here, the zygomorphic flowers show a particularly extensive receptacular growth, resulting in the formation of a spur-like receptacular cavity ('inner spur'). The nectary gland is hidden at the base of the cavity. Various forms of compartmentalization, culminating in the 'revolver flower' of Geranium maderense, are described. Conclusions: Despite the superficial similarity of the flowers in Geraniaceae and Hypseocharitaceae, there is broad diversity in floral organization and floral architecture. While the receptacular origin of the spur-like cavity in Pelargonium had already been described, anthophore formation via intercalary growth of the receptacle in the other genera had not been previously documented. In the context of the most recent phylogenies of the families, an evolutionary series for the floral architecture is proposed, underscoring the importance of synorganization in these seemingly simple flowers.

Dispersing towards Madagascar: Biogeography and evolution of the Madagascan endemics of the Spermacoceae tribe (Rubiaceae).

Despite the close proximity of the African mainland, dispersal of plant lineages towards Madagascar remains intriguing. The composition of the Madagascan flora is rather mixed and shows besides African representatives, also floral elements of India, Southeast Asia, Australia, and the Neotropics. Due to its proportionally large number of Madagascan endemics, the taxonomically troublesome Spermacoceae tribe is an interesting group to investigate the origin and evolution of the herbaceous Rubiaceae endemic to Madagascar. The phylogenetic position of these endemics were inferred using four plastid gene markers. Age estimates were obtained by expanding the Spermacoceae dataset with representatives of all Rubiaceae tribes. This allowed incorporation of multiple fossil-based calibration points from the Rubiaceae fossil record. Despite the high morphological diversity of the endemic herbaceous Spermacoceae on Madagascar, only two colonization events gave rise to their current diversity. The first clade contains Lathraeocarpa, Phylohydrax and Gomphocalyx, whereas the second Madagascan clade includes the endemic genera Astiella, Phialiphora, Thamnoldenlandia and Amphistemon. The tribe Spermacoceae is estimated to have a Late Eocene origin, and diversified during Oligocene and Miocene. The two Madagascan clades of the tribe originated in the Oligocene and radiated in the Miocene. The origin of the Madagascan Spermacoceae cannot be explained by Gondwanan vicariance but only by means of Cenozoic long distance dispersal events. Interestingly, not only colonization from Africa occurred but also long distance dispersal from the Neotropics shaped the current diversity of the Spermacoceae tribe on Madagascar.

Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae.

BACKGROUND AND AIMS: The first documented observation of pollination in Pleurothallidinae was that of Endrés, who noticed that the 'viscid sepals' of Specklinia endotrachys were visited by a 'small fly'. Chase would later identify the visiting flies as being members of the genus Drosophila. This study documents and describes how species of the S. endotrachys complex are pollinated by different Drosophila species. METHODS: Specimens of Specklinia and Drosophila were collected in the field in Costa Rica and preserved in the JBL and L herbaria. Flies were photographed, filmed and observed for several days during a 2-year period and were identified by a combination of non-invasive DNA barcoding and anatomical surveys. Tissue samples of the sepals, petals and labellum of Specklinia species were observed and documented by SEM, LM and TEM. Electroantennogram experiments were carried out on Drosophila hydei using the known aggregation pheromones ethyl tiglate, methyl tiglate and isopropyl tiglate. Floral compounds were analysed by gas chromatography-mass spectometry using those same pheromones as standards. KEY RESULTS: Flowers of S. endotrachys, S. pfavii, S. remotiflora and S. spectabilis are visited and pollinated by several different but closely related Drosophila species. The flies are arrested by aggregation pheromones, including ethyl tiglate, methyl tiglate and isopropyl tiglate, released by the flowers, and to which at least D. hydei is very sensitive. Visible nectar drops on the adaxial surface of sepals are secreted by nectar-secreting stomata, encouraging male and female Drosophila to linger on the flowers for several hours at a time. The flies frequently show courtship behaviour, occasionally copulating. Several different Drosophila species can be found on a single Specklinia species. CONCLUSIONS: Species of the S. endotrachys group share a similar pollination syndrome. There seem to be no species-specific relationships between the orchids and the flies. It is not expected that Specklinia species will hybridize naturally as their populations do not overlap geographically. The combination of pheromone attraction and nectar feeding is likely to be a generalized pollination syndrome in Pleurothallidinae.

Evolution of mycoheterotrophy in Polygalaceae: The case of Epirixanthes.

PREMISE OF THE STUDY: The mycoheterotrophic lifestyle has enabled some plant lineages to obtain carbon from their mycorrhizal symbionts. The mycoheterotrophic genus Epirixanthes (Polygalaceae) consists of six species from tropical Asia. Although it is probably closely related to the chlorophyllous genus Salomonia and linked to arbuscular mycorrhizal fungi, lack of DNA sequence data has thus far prevented these hypotheses from being tested. Therefore, the evolutionary history of Epirixanthes remains largely unknown. METHODS: We reconstructed the phylogenetic relationships of Epirixanthes based on nuclear ITS and plastid matK data. Divergence times were inferred using a Bayesian relaxed clock approach, and we phylogenetically analyzed its mycorrhizal symbionts. We furthermore assigned these symbionts to operational taxonomic units, compared them with symbionts of other Polygalaceae, and measured their phylogenetic diversity. KEY RESULTS: We found that Epirixanthes is placed in tribe Polygaleae as sister to Salomonia. Epirixanthes has a Miocene-Oligocene stem age and grows exclusively in symbiosis with fungi of Glomeraceae. Salomonia and some Polygala species are linked to both Glomeraceae and Acaulosporaceae, resulting in higher phylogenetic diversity values. The majority of the symbionts of Epirixanthes are not found in Salomonia or Polygala, although a few shared fungal taxa are found. CONCLUSIONS: Epirixanthes forms a relatively young mycoheterotrophic lineage. The Oligocene-Miocene origin suggests its evolution was influenced by the environmental dynamics in Southeast Asia during this time. Although comparison of fungi from Epirixanthes with those from Salomonia and Polygala suggests some specialization, many other mycoheterotrophic plants are linked to a more narrow set of Glomeraceae.

A plastid DNA phylogeny of tribe Miliuseae: insights into relationships and character evolution in one of the most recalcitrant major clades of Annonaceae.

PREMISE OF THE STUDY: Tribe Miliuseae (∼25 genera and ∼510 species) includes a substantial part of the species and generic diversity in the pantropical flowering-plant family Annonaceae (∼108 genera and ∼2400 species). Previous molecular phylogenetic analyses have failed to resolve the backbone phylogeny of the tribe, impeding biogeographical and evolutionary studies. We use a dense generic taxon sample (∼89% of generic diversity in Miliuseae) and plastid DNA sequence data (∼7 kb) to clarify the phylogenetic relationships of and within the tribe. METHODS: Parsimony and Bayesian phylogenetic reconstructions and ancestral character-state reconstructions of several reproductive characters were performed. KEY RESULTS: Dendrokingstoniae, Monocarpieae, and Miliuseae are recovered in a strongly supported clade, and each tribe is strongly supported as monophyletic. Miliuseae are characterized by a synapomorphic cryptoaperturate/disulculate pollen apertural system. Stenanona is shown to be nested within the paraphyletic genus Desmopsis. The only Neotropical clade (Sapranthus, Tridimeris, Desmopsis, and Stenanona) in the predominantly Asian Miliuseae is shown to be closely related to an undescribed genus from continental Southeast Asia and the Indo-Malayan and Austral-Pacific genus Meiogyne. Ancestral character-state reconstructions of several reproductive characters that are diagnostically important at the generic level indicate a considerable degree of homoplasy. CONCLUSIONS: The results improve our understanding of the relationships of and within Miliuseae, but parts of the backbone of the phylogeny remain poorly supported. Additional data from variable nuclear markers or reduced-genome-representation approaches seem to be required to further resolve relationships within this recalcitrant clade.

New insights in the long-debated evolutionary history of Triuridaceae (Pandanales).

The mycoheterotrophic plant family Triuridaceae (Pandanales) is hypothesized to be an old family, mainly based on its pantropical distribution. The existence of fossils from the Upper Cretaceous, assigned to Triuridaceae may form additional support for a great age of the family, although the affinity of these fossils to Triuridaceae is questioned. Although the circumscription of Triuridaceae has never been problematic, probably due to its distinct morphological characters, its systematic relationship has been under debate since the family was described around 1840. The lack of synapomorphies suitable for resolving higher taxonomic relationships is a function of the family's reduced vegetative growth and the highly modified floral structures. Molecular studies have assigned Triuridaceae to Pandanales, but its exact phylogenetic position remains unknown. In the present study the phylogeny of the Pandanales was reconstructed using four molecular markers and the divergence age estimates were obtained with a relaxed molecular clock method. We found that Triuridaceae are monophyletic and most likely descent form the second major split in Pandanales. The relationships between the other Pandanales families (Cyclanthaceae, Pandanaceae, Stemonaceae and Velloziaceae) are otherwise in accordance with earlier studies. Velloziaceae are sister to the rest of the Pandanales, Stemonaceae are most likely sister to a clade consisting of Pandanaceae and Cyclanthaceae, and the latter two families are sister to each other. All currently recognized tribes within Triuridaceae are also monophyletic at current taxon sampling. We estimate that the family has a Cretaceous (or Lower Paleocene) stem age, which is in accordance with earlier predictions. This old age, along with elevated mutation rates indicated by long branch lengths and the family's mycoheterotrophic lifestyle, might account for the substantial morphological differences between Triuridaceae and its closest relatives.

Phylogenetic relationships of the mycoheterotrophic genus Voyria and the implications for the biogeographic history of Gentianaceae.

PREMISE OF THE STUDY: The angiosperm family Gentianaceae comprises over 1700 species in 91 genera. Gentianaceae are distributed worldwide, but most species occur in temperate zones. Phylogenetic studies demonstrate that the family consists of six monophyletic tribes. However, the phylogenetic position of the mycoheterotrophic genus Voyria, with a remarkable trans-Atlantic distribution, remained unknown. METHODS: We used nuclear ITS and 18S rDNA and mitochondrial apt1 and matR data to infer the phylogenetic position of Voyria in Gentianaceae. In addition, with Bayesian relaxed molecular clock analyses we obtained age estimates for the diversification of Voyria and Gentianaceae in general and used these results to reconstruct the ancestral areas associated with the early diversification events in Gentianaceae. KEY RESULTS: Our results demonstrate that Voyria is an early diverging lineage within Gentianaceae with no close relationships to other mycoheterotrophic Gentianaceae lineages. Voyria originated in the neotropics during the Early Eocene but only reached its current transoceanic distribution around the end of the Oligocene. The neotropics were an important area for the early diversification events in Gentianaceae, most of which occurred during the Eocene. CONCLUSIONS: Voyria is an old, phylogenetically isolated lineage within Gentianaceae, and the current distribution of the genus is indicative of the ancestral area in which the early diversification events of Gentianaceae occurred. In parallel with many other pantropical families, our results suggest that migration of tropical taxa through Laurasia during the Early Eocene has played an important role in shaping the current global distribution of Gentianaceae.

Orchids reduce attachment of herbivorous snails with leaf trichomes.

Protective structures in the epidermis are essential for land plants to defend themselves against herbivores. In this study, we investigated the effect of different types of trichomes of three orchids, Calanthe triplicata, Dendrochilum pallidiflavens and Trichotosia ferox, on attachment of herbivorous land snails, using histochemistry and centrifuge experiments. Size, ornamentation and histochemistry of epicuticular trichomes on the orchid leaves were assessed with light microscopy, scanning electron microscopy and transmission electron microscopy. Total forces needed to detach two differently shaped snail species, Subulina octona and Pleurodonte isabella, were measured using a turntable equipped with a synchronized strobe. Snails were placed in two positions, either perpendicular or parallel to the main veins on the orchid leaves, both on the adaxial (= upper) or abaxial (= lower) side. The results obtained provided three new insights. First, a perpendicular or parallel position of the snails to the main veins did not significantly affect the attachment performance of either species tested. Secondly, snails detached significantly easier on leaf sides covered with a high density of lignin filled epicuticular trichomes. Thirdly, the removal of glandular trichomes did not affect the attachment forces; however, the absence of lignified trichomes increased the attachment of the snails. Our study highlights the importance of studying micro-ornamentation in combination with performance for obtaining a better understanding of the defense mechanisms employed by different species of orchids to deter herbivorous snails.

Mycoheterotrophic interactions are not limited to a narrow phylogenetic range of arbuscular mycorrhizal fungi.

The majority of achlorophyllous mycoheterotrophic plant species associate with arbuscular mycorrhizal fungi (AMF). Previous studies have shown that some species are highly specialized towards narrow lineages of AMF and have suggested that only particular lineages of these fungi are targeted by mycoheterotrophic plants. To test this hypothesis, we analyzed all available partial SSU sequences of AMF associated with mycoheterotrophic plants including data from 13 additional specimens from French Guiana, Gabon and Australia. Sequences were assigned to 'virtual taxa' (VT) according to the MaarjAM database. We found that 20% of all known Glomeromycota VT are involved in mycoheterotrophic interactions and the majority of associations involve Glomeraceae (Glomus Group A) fungi. While some mycoheterotrophic plant species have been found growing with only a single VT, many species are able to associate with a wide range of AMF. We calculated significant phylogenetic clustering of Glomeromycota VT involved in mycoheterotrophic interactions, suggesting that associations between mycoheterotrophic plants and AMF are influenced by the phylogenetic relationships of the fungi. Our results demonstrate that many lineages of AMF are prone to exploitation by mycoheterotrophic plants. However, mycoheterotrophs from different plant lineages and different geographical regions tend to be dependent on lineages of AMF that are phylogenetically related.

Floral development of Hydrocera and Impatiens reveals evolutionary trends in the most early diverged lineages of the Balsaminaceae.

BACKGROUND AND AIMS: Balsaminaceae consist of two genera, the monospecific Hydrocera and its species-rich sister Impatiens. Although both genera are seemingly rather similar in overall appearance, they differ in ecology, distribution range, habitat preference and morphology. Because morphological support for the current molecular phylogenetic hypothesis of Impatiens is low, a developmental study is necessary in order to obtain better insights into the evolutionary history of the family. Therefore, the floral development of H. triflora and I. omeiana was investigated, representing the most early-diverged lineage of Impatiens, and the observations were compared with the literature. METHODS: Flowers at all developmental stages were examined using scanning electron microscopy and light microscopy. KEY RESULTS: In Hydrocera, two whorls of five free perianth primordia develop into a less zygomorphic perianth compared with its sister genus. The androecial cap originates from five individual stamen primordia. Post-genital fusion of the upper parts of the filaments result in a filament ring below the anthers. The anthers fuse forming connivent anther-like units. The gynoecium of Hydrocera is pentamerous; it is largely synascidiate in early development. Only then is a symplicate zone formed resulting in style and stigmas. In I. omeiana, the perianth is formed as in Hydrocera. Five individual stamen primordia develop into five stamens, of which the UPPER PART OF THE FILAMENTS CONVERGE WITH EACH OTHER. THE GYNOECIUM OF I. OMEIANA IS TETRAMEROUS; IT APPEARS ANNULAR IN EARLY DEVELOPMENT. CONCLUSIONS: Comparison of the present results with developmental data from the literature confirms the perianth morphocline hypothesis in which a congenital fusion of the parts of the perianth results in a shift from pentasepalous to trisepalous flowers. In addition, the development of the androecial cap and the gynoecium follows several distinct ontogenetic sequences within the family.

Biodiversity priority areas and conservation strategies for seed plants in China.

China is known for its abundant plant resources, but biodiversity conservation faces unprecedented challenges. To provide feasible suggestions for sustainable conservation, we used the species richness algorithm and complementary algorithm to study distribution patterns of 34,082 seed plants based on 1,007,196 county-level distribution records. We reconstructed a phylogenetic tree for 95.35% of species and estimated the spatial phylogenetics, followed by correlation analyses between different distribution patterns. We identified 264 counties concentrated in southern and south-western mountainous areas as hotspots which covered 10% of the land area of China and harbored 85.22% of the Chinese seed plant species. The biodiversity conservation priorities we identified were highly representative as we have considered multiple conservation indicators. We evaluated the conservation effectiveness and gaps in the network of nature reserves and identified 31.44, 32.95, and 9.47%, respectively, of the hotspot counties as gaps in the national nature reserves, provincial nature reserves and both together, with respectively 55.77, 61.53, and 28.94% of the species. Analysis of the species composition showed there were a large number of threatened and endemic species occurring in the nature reserves' gaps. The conservation gaps need to be filled by establishing new nature reserves or national parks, especially in south-western China, and more attentions should be paid to strengthen the conservation of specific plant taxa due to the apparent mismatches between different distribution patterns.

Antimicrobial Activity of Necklace Orchids is Phylogenetically Clustered and can be Predicted With a Biological Response Method.

Necklace orchids (Coelogyninae, Epidendroideae) have been used in traditional medicine practices for centuries. Previous studies on a subset of unrelated orchid species utilized in these traditional practices revealed they possessed antimicrobial, anti-inflammatory, and anti-oxidant activity, providing experimental proof for their medicinal properties. To date however none of these species have been investigated ethno-botanically in a phylogenetic context. This study carried out comparative bioprospecting for a group of wild orchids using EBDCS (the Economic Botany Data Collection Standards) organ targeted and biological response methods. The traditional medicinal use of necklace orchids was recorded from books and journals published between 1984 and 2016. Two orchids, Coelogyne cristata and Coelogyne fimbriata, were selected, cultivated both indoors and outdoors, and the antimicrobial properties on extracts from their leaves and pseudobulbs tested against a selection of human pathogens. A molecular phylogeny of Coelogyninae based on nuclear ribosomal ITS and plastid matK DNA sequences obtained from 148 species was reconstructed with Maximum Likelihood (ML) using RAxML, Maximum Parsimony (MP) using PAUP and Bayesian Inference using MrBayes. Bioprospecting comparison of EBDCS and biological response was carried out using customized R scripts. Ethanolic extracts obtained from leaves of C. fimbriata inhibited growth of Bacillus cereus, Staphylococcus aureus, and Yersinia enterocolitica, confirming the antimicrobial properties of these extracts. Leaf extracts were found to have slightly stronger antimicrobial properties for plants cultivated outdoors than indoors. These differences were not found to be statistically significant though. Three hot nodes with high potency for antimicrobial activities were detected with the EBDCS organ targeted classification method, and eight hot nodes were detected with the biological response classification method. The biological response classification method is thus a more effective tool in finding hot nodes amongst clades of species with high medicinal potential.

Rapid radiation of Impatiens (Balsaminaceae) during Pliocene and Pleistocene: result of a global climate change.

Impatiens comprises more than 1000 species and is one of the largest genera of flowering plants. The genus has a subcosmopolitan distribution, yet most of its evolutionary history is unknown. Diversification analyses, divergence time estimates and historical biogeography, illustrated that the extant species of Impatiens originated in Southwest China and started to diversify in the Early Miocene. Until the Early Pliocene, the net diversification rate within the genus was fairly slow. Since that time, however, approximately 80% of all Impatiens lineages have originated. This period of rapid diversification coincides with the global cooling of the Earth's climate and subsequent glacial oscillations. Without this accelerated diversification rate, Impatiens would only have contained 1/5th of its current number of species, thereby indicating the rapid radiation of the genus.

Morphological and Molecular Characterization of Orchid Fruit Development.

Efficient seed dispersal in flowering plants is enabled by the development of fruits, which can be either dehiscent or indehiscent. Dehiscent fruits open at maturity to shatter the seeds, while indehiscent fruits do not open and the seeds are dispersed in various ways. The diversity in fruit morphology and seed shattering mechanisms is enormous within the flowering plants. How these different fruit types develop and which molecular networks are driving fruit diversification is still largely unknown, despite progress in eudicot model species. The orchid family, known for its astonishing floral diversity, displays a huge variation in fruit dehiscence types, which have been poorly investigated. We undertook a combined approach to understand fruit morphology and dehiscence in different orchid species to get more insight into the molecular network that underlies orchid fruit development. We describe fruit development in detail for the epiphytic orchid species Erycina pusilla and compare it to two terrestrial orchid species: Cynorkis fastigiata and Epipactis helleborine. Our anatomical analysis provides further evidence for the split carpel model, which explains the presence of three fertile and three sterile valves in most orchid species. Interesting differences were observed in the lignification patterns of the dehiscence zones. While C. fastigiata and E. helleborine develop a lignified layer at the valve boundaries, E. pusilla fruits did not lignify at these boundaries, but formed a cuticle-like layer instead. We characterized orthologs of fruit-associated MADS-domain transcription factors and of the Arabidopsis dehiscence-related genes INDEHISCENT (IND)/HECATE 3 (HEC3), REPLUMLESS (RPL) and SPATULA (SPT)/ALCATRAZ (ALC) in E. pusilla, and found that the key players of the eudicot fruit regulatory network appear well-conserved in monocots. Protein-protein interaction studies revealed that MADS-domain complexes comprised of FRUITFULL (FUL), SEPALLATA (SEP) and AGAMOUS (AG) /SHATTERPROOF (SHP) orthologs can also be formed in E. pusilla, and that the expression of HEC3, RPL, and SPT can be associated with dehiscence zone development similar to Arabidopsis. Our expression analysis also indicates differences, however, which may underlie fruit divergence.

Diversification of myco-heterotrophic angiosperms: evidence from Burmanniaceae.

BACKGROUND: Myco-heterotrophy evolved independently several times during angiosperm evolution. Although many species of myco-heterotrophic plants are highly endemic and long-distance dispersal seems unlikely, some genera are widely dispersed and have pantropical distributions, often with large disjunctions. Traditionally this has been interpreted as evidence for an old age of these taxa. However, due to their scarcity and highly reduced plastid genomes our understanding about the evolutionary histories of the angiosperm myco-heterotrophic groups is poor. RESULTS: We provide a hypothesis for the diversification of the myco-heterotrophic family Burmanniaceae. Phylogenetic inference, combined with biogeographical analyses, molecular divergence time estimates, and diversification analyses suggest that Burmanniaceae originated in West Gondwana and started to diversify during the Late Cretaceous. Diversification and migration of the species-rich pantropical genera Burmannia and Gymnosiphon display congruent patterns. Diversification began during the Eocene, when global temperatures peaked and tropical forests occurred at low latitudes. Simultaneous migration from the New to the Old World in Burmannia and Gymnosiphon occurred via boreotropical migration routes. Subsequent Oligocene cooling and breakup of boreotropical flora ended New-Old World migration and caused a gradual decrease in diversification rate in Burmanniaceae. CONCLUSION: Our results indicate that extant diversity and pantropical distribution of myco-heterotrophic Burmanniaceae is the result of diversification and boreotropical migration during the Eocene when tropical rain forest expanded dramatically.

Selection on length mutations after frameshift can explain the origin and retention of the AP3/DEF-like paralogues in Impatiens.

Evolution of class B genes through gene duplication has been proposed as an evolutionary mechanism that contributed to the enormous floral diversity. Frameshift mutations are a likely mechanism to explain the divergent C-terminal sequences of MIKC gene subfamilies. So far, the inferences for frameshifts and selective pressures on the C-terminal domain are made for old duplications for which the exact selective pressures are obscured by evolutionary time. This motivated us to study an example of a recent duplication, which allows us to consider in more detail the selective pressures that are involved after duplication. We find that after duplication and frameshift of Impatiens class B genes, the individual codons show no evidence for adaptive selection. It is rather the length of the C-terminal domain that either is strictly conserved or varies strongly. This suggests a role for the length of the C-terminal domain in the retention of duplicated genes.

Elaborate petals in Australian Spermacoce (Rubiaceae) species: morphology, ontogeny and function.

BACKGROUND AND AIMS: Australian Spermacoce species display various types of elaborate petals. Their precise morphology, ontogenetic origin, and function are hitherto unknown. The aim of the present paper is to unravel the development and nature of the diverse types of elaborate petals in Spermacoce through a floral ontogenetic study. METHODS: The floral ontogeny of six species characterized by different types of corolla appendages was studied by scanning electron microscopy and light microscopy. In order to elucidate the possible functions of the elaborate petals, field observations were conducted as well. KEY RESULTS: Scanning electronmicrographs show that full-grown petals of Spermacoce lignosa, S. phaeosperma and S. redacta bear appendages on their ventral side. Despite their different appearance at anthesis, the appendages develop very similarly in all three species. They are initiated at the same developmental stage and are first visible as two arcs of primordia converging from the upper margins of the petal towards its midvein and downwards. In S. brevidens, S. caudata and S. erectiloba, the full-grown petals have two long, concave protuberances, which develop from the tissue at both sides of the petal's mid-vein. In these three species, initiation of appendages on the ventral side of the petals is also observed, but they are hardly visible on the mature petals. The two types of elaborate petals tightly enclose the anthers, both in bud and during most of the flowering period. CONCLUSIONS: Among Australian Spermacoce species, two types of elaborate petals can be distinguished. The former hypothesis that the two types of elaborate petals are essentially homologous is here rejected. Field investigations point out that the elaborate petals might play a role in the pollination biology of the species.

A floral ontogenetic study on the sister group relationship between the genus Samolus (Primulaceae) and the Theophrastaceae.

The former Primulales used to be subdivided into the woody Theophrastaceae and Myrsinaceae, from the tropics and subtropics, and the herbaceous Primulaceae, which are mainly found in the temperate regions of the northern hemisphere. Recent analyses based on morphological as well as molecular data revealed a close relationship between the genus Samolus L. of Primulaceae and the monophyletic family Theophrastaceae. We studied the floral development of six species from four different genera of Theophrastaceae and compared it to floral ontogenetical data of Samolus valerandi L. to find support for a close relationship. Samolus and the members of Theophrastaceae share the presence of staminodes and a similar development of the placenta and the ovules. Apart from the different habit and distribution, however, we also observed some major differences between both lineages, such as the absence of common primordia in Theophrastaceae, the development of a gynoecial cap in Samolus, and the difference in development, shape, and structure of the staminodes. Therefore, we propose to keep Samolus separated from the genera of the Theophrastaceae, and we suggest that it be raised to family level.