Molecular and morphological phylogenetics of the digitate-tubered clade within subtribe Orchidinae s.s. (Orchidaceae: Orchideae)

The digitate-tubered clade (Dactylorhiza s.l. plus Gymnadenia s.l.) within subtribe Orchidinae is an important element of the North-temperate orchid flora and has become a model system for studying the genetic and epigenetic consequences of organism-wide ploidy change. Here, we integrate morphological phylogenetics with Sanger sequencing of nrITS and the plastid region trnL-F in order to explore phylogenetic relationships and phenotypic character evolution within the clade. The resulting morphological phylogenies are strongly incongruent with the molecular phylogenies, instead reconstructing through parsimony the genus-level boundaries recognised by traditional 20th Century taxonomy. They raise fresh doubts concerning whether Pseudorchis is sister to Platanthera or to Dactylorhiza plus Gymnadenia. Constraining the morphological matrix to the topology derived from ITS sequences increased tree length by 20%, adding considerably to the already exceptional level of phenotypic homoplasy. Both molecular and morphological trees agree that D. viridis and D. iberica are the earliest- diverging species within Dactylorhiza (emphasising the redundancy of the former genus Coeloglossum). Morphology and ITS both suggest that the former genus Nigritella is nested within (and thus part of) Gymnadenia, the Pyrenean endemic 'N.' gabasiana apparently forming a molecular bridge between the two radically contrasting core phenotypes. Comparatively short subtending molecular branches plus widespread (though sporadic) hybridisation indicate that Dactylorhiza and Gymnadenia approximate the minimum level of molecular divergence acceptable in sister genera. They share similar tuber morphologies and base chromosome numbers, and both genera are unusually prone to polyploid speciation. Another prominent feature of multiple speciation events within Gymnadenia is floral paedomorphosis. The 'traditional' morphological and candidate-gene approaches to phylogeny reconstruction are critically appraised.Peer reviewedFinal Published versio

The Good, The Bad, The Ugly: Using Naturally Occurring Terata to Distinguish the Possible from the Impossible in Orchid Floral Evolution

We interpret extensive field observations of terata in the context of recent insights into monocot phylogeny and evolutionary-developmental genetics to explore the evolution of the orchid flower. Our arguably typological classification of floral terata focuses on natural occurrences of three contrasting modes of peloria (restoration of actinomorphy in a formerly zygomorphic perianth) and three contrasting modes of pseudopeloria (lessening of the degree of zygomorphy shown by the evolutionarily preceding perianth). Dynamic evolutionary transitions in floral morphology are assigned to recently revised concepts of heterotopy (including homeosis: evolutionary transitions in position of expression) and heterochrony (evolutionary transitions in timing of expression), seeking patterns that delimit developmental constraints and allow inferences regarding underlying genetic controls. Lateral heterotopy, occurring within the whorl of three petals (including the labellum) or within the adjacent whorl of three sepals, is more frequent than acropetal heterotopy, and full basipetal heterotopy does not occur. Pseudopeloria is more likely than peloria to generate a radically altered yet functional perianth but is also more likely to cause acropetal modification of the fused filaments and style that constitute the characteristic gynostemium of orchids. We infer that at least one gene or gene complex controls stylestamen fusion, which requires the preadaptation of strongly developed epigyny, and another determines both stamen suppression and labellum formation adaxially. Our earlier hypothesis implicating genes of the TCP family has recently been challenged by empirical evidence of complex interactions between several MADS-box genes. Many transitions are highly iterative, and some are reversible (atavistic). Once heritability has been demonstrated, the most effective criteria for determining the most appropriate taxonomic status of a novel morph are the profundity of the phenotypic shift that it represents, the number and uniformity of the resulting populations, and whether the novel morph subsequently diversified to generate further morphs that retain the innovative features. Although morphological transitions attributable to heterochrony may be a more common driver of speciation than those attributable to heterotopy, we demonstrate that arguably all of the modes of instantaneous floral transition described in this paper have the ability to generate prospecies

The Operculum in Pollen of Monocotyledons

Within monocotyledons, monosulcate pollen is the predominant type and probably represents the plesiomorphic condition, but considerable variation occurs in sulcus morphology. An operculum is an exine thickening that covers most of an aperture. Monocot opercula are usually associated with sulci, although they can occur in ulcerate apertures, as in Poaceae. There are several other aperture types closely related to the monosulcate-operculate type, and confusion occurs in the palynological literature between monosulcate-operculate, pontoperculate, disulculate, disulcate, and zona-aperturate (zonasulculate or zonasulcate) pollen. Transmission electron microscopy (TEM) was used to determine the distribution of the thick apertural intine and to accurately identify these aperture types. Operculate pollen most frequently was present in Asparagales (particularly Agavaceae, Doryanthaceae, lridaceae, and Tecophilaeaceae), Liliales (particularly Liliaceae, Melanthiaceae, and Uvulariaceae), and relatively infrequently among commelinid monocots, except for some Arecaceae, Dasypogonaceae, and Poales. Thus, we conclude that opercula have probably evolved several times independently within monocots, particularly in taxa from dry or seasonally dry habitats, and that this adaptation may be related to their function in protecting the aperture. Two transformation series of related aperture types are proposed, one of which involves monosulcate-operculate pollen, although further testing will be required

Patterns of Floral Structure and Orientation in Japonolirion , Narthecium, and Tofieldia

Floral evolution requires reassessment in basal monocots, including species formerly assigned to Melanthiaceae, in the light of recent developments in the molecular phylogenetics of monocots. We have investigated flowers of Tofieldia (Tofieldiaceae), Japonolirion (Petrosaviaceae), and Narthecium (Nartheciaceae). We confirm Engler\u27s (1888) hypothesis that orientation of lateral flowers in monocots is dependent on presence and position of additional phyllomes on the pedicel. The type of floral orientation that occurs in Tofieldia is unusual for monocots, since the additional phyllomes are represented by calyculus scales rather than a bracteole, and the outer whorl tepals are initiated alternating with the calyculus scales. In Japonolirion and Narthecium, a bracteole is inserted in an adaxialtransverse or transverse position; either the outer median tepal is adaxial or no single tepal is inserted in the median position. In Tofieldia, the pedicel has a calyculus of an abaxial and two adaxialtransverse phyllomes; the outer median tepal is adaxial. Additional phyllomes on the pedicel are not adaxial, in contrast to adaxial prophylls in the vegetative regions. The presence or absence of a bracteole or calyculus is taxonomically important. Tofieldia pusilla differs from the other species of Tofieldia examined in the absence of a flower-subtending bract, but the calyculus demonstrates some bract-like features in position, structure and development, which can be interpreted as a hybridization of developmental pathways. The abaxial calyculus scale of T. coccinea is delayed in development

Molecular Studies of Subfamily Gilliesioideae (Alliaceae)

We present an analysis of relationships in Gilliesioideae (Alliaceae) based on a combined matrix of plastid rbcL, the trnL intron, the trnL-F intergenic spacer, and the rps16 intron and nuclear ITS ribosomal DNA sequences. The results are generally congruent with previous analyses, indicating two well-supported groups: Ipheion plus allied genera ( lpheieae ined.) and Gilliesieae. They also provide higher bootstrap support for many patterns of relationships. Polyphyly of lpheion and Nothoscordum is confirmed. Increased taxon sampling (particularly in Gilliesieae) and additional molecular data would be desirable to provide further resolution and to allow an appropriate taxonomic revision to be made

Flower-specific KNOX phenotype in the orchid Dactylorhiza fuchsii

The KNOTTED1-like homeobox (KNOX) genes are best known for maintaining a pluripotent stem-cell population in the shoot apical meristem that underlies indeterminate vegetative growth, allowing plants to adapt their development to suit the prevailing environmental conditions. More recently, the function of the KNOXgene family has been expanded to include additional roles in lateral organ development such as complex leaf morphogenesis, which has come to dominate the KNOX literature. Despite several reports implicating KNOX genes in the development of carpels and floral elaborations such as petal spurs, few authors have investigated the role of KNOX genes in flower development. Evidence is presented here of a flower-specific KNOX function in the development of the elaborate flowers of the orchid Dactylorhiza fuchsii, which have a three-lobed labellum petal with a prominent spur. Using degenerate PCR, four Class I KNOX genes (DfKN1–4) have been isolated, one from each of the four major Class I KNOX subclades and by reverse transcription PCR (RT-PCR), it is demonstrated that DfKNOXtranscripts are detectable in developing floral organs such as the spur-bearing labellum and inferior ovary. Although constitutive expression of the DfKN2 transcript in tobacco produces a wide range of floral abnormalities, including serrated petal margins, extra petal tissue, and fused organs, none of the vegetative phenotypes typical of constitutive KNOX expression were produced. These data are highly suggestive of a role for KNOX expression in floral development that may be especially important in taxa with elaborate flowers

A taxonomic revision of the myrmecophilous species of the rattan genus Korthalsia (Arecaceae)

The rattan genus Korthalsia Blume (Arecaceae: Calamoideae: Calameae) is widespread in the Malesian region. Among the 28 accepted species are 10 species that form intimate associations with ants. The ants inhabit the conspicuous ocreas that are produced by these species, using them as domatia to care for their young and to husband scale insects. As a foundation for future work, we present here a taxonomic treatment of the myrmecophilous Korthalsia species, based on extensive research both in the herbarium and the field. In addition, we conduct detailed morphological characterisation of the structure and development of ocrea using light and scanning electron microscopy. Descriptions, illustrations, keys and distribution maps are presented for all 10 species, along with microscopic images of ocrea morphology and development for selected species

Ultrastructure and optics of the prism-like petal epidermal cells of Eschscholzia californica (California poppy).

The petals of Eschscholzia californica (California poppy) are robust, pliable and typically coloured intensely orange or yellow owing to the presence of carotenoid pigments; they are also highly reflective at certain angles, producing a silky effect. To understand the mechanisms behind colour enhancement and reflectivity in California poppy, which represents a model species among early-divergent eudicots, we explored the development, ultrastructure, pigment composition and optical properties of the petals using light microscopy and electron microscopy combined with both spectrophotometry and goniometry. The elongated petal epidermal cells each possess a densely thickened prism-like ridge that is composed primarily of cell wall. The surface ridges strongly focus incident light onto the pigments, which are located in plastids at the cell base. Our results indicate that this highly unusual, deeply ridged surface structure not only enhances the deep colour response in this desert species, but also results in strongly angle-dependent 'silky' reflectivity that is anisotropic and mostly directional

Structural colour from helicoidal cell-wall architecture in fruits of Margaritaria nobilis

The bright and intense blue-green coloration of the fruits of Margaritaria nobilis (Phyllanthaceae) was investigated using polarization-resolved spectroscopy and transmission electron microscopy. Optical measurements of freshly collected fruits revealed a strong circularly polarized reflection of the fruit that originates from a cellulose helicoidal cell wall structure in the pericarp cells. Hyperspectral microscopy was used to capture the iridescent effect at the single-cell level.Leverhulme Trust (F/09-741/G)United States. Air Force Office of Scientific Research (award number FA9550-10-1-0020)Adolphe Merkle FoundationSwiss National Science Foundation (National Centre of Competence in Research Bio-Inspired Materials)Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC David Phillips fellowship (BB/K014617/1)

Structural colour in Chondrus crispus.

The marine world is incredibly rich in brilliant and intense colours. Photonic structures are found in many different species and provide extremely complex optical responses that cannot be achieved solely by pigments. In this study we examine the cuticular structure of the red alga Chondrus crispus (Irish Moss) using anatomical and optical approaches. We experimentally measure the optical response of the multilayer structure in the cuticle. Using finite-difference time-domain modelling, we demonstrate conclusively for the first time that the dimensions and organisation of lamellae are responsible for the blue structural colouration on the surface of the fronds. Comparison of material along the apical-basal axis of the frond demonstrates that structural colour is confined to the tips of the thalli and show definitively that a lack of structural colour elsewhere corresponds with a reduction in the number of lamellae and the regularity of their ordering. Moreover, by studying the optical response for different hydration conditions, we demonstrate that the cuticular structure is highly porous and that the presence of water plays a critical role in its ability to act as a structural light reflector.The research leading to these results has received funding from the BBSRC David Phillips fellowship (BBSRC David Phillips, BB/K014617/1). BJG thanks the Leverhulme Trust grant (F/09-741/G). RHW thanks the British Phycological Society for a Project Award (2012).This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/srep1164