The evolution of the duckweed ionome mirrors losses in structural complexity.

BACKGROUND AND AIMS: The duckweeds (Lemnaceae) consist of 36 species exhibiting impressive phenotypic variation, including the progressive evolutionary loss of a fundamental plant organ, the root. Loss of roots and reduction of vascular tissues in recently derived taxa occur in concert with genome expansions of ≤14-fold. Given the paired loss of roots and reduction in structural complexity in derived taxa, we focus on the evolution of the ionome (whole-plant elemental contents) in the context of these fundamental changes in body plan. We expect that progressive vestigiality and eventual loss of roots might have both adaptive and maladaptive consequences that are hitherto unknown. METHODS: We quantified the ionomes of 34 accessions in 21 species across all duckweed genera, spanning 70 Myr in this rapidly cycling plant (doubling times are as rapid as ~24 h). We related both micro- and macroevolutionary ionome contrasts to body plan remodelling and showed nimble microevolutionary shifts in elemental accumulation and exclusion in novel accessions. KEY RESULTS: We observed a robust directional trend in calcium and magnesium levels, decreasing from the ancestral representative Spirodela genus towards the derived rootless Wolffia, with the latter also accumulating cadmium. We also identified abundant within-species variation and hyperaccumulators of specific elements, with this extensive variation at the fine (as opposed to broad) scale. CONCLUSIONS: These data underscore the impact of root loss and reveal the very fine scale of microevolutionary variation in hyperaccumulation and exclusion of a wide range of elements. Broadly, they might point to trade-offs not well recognized in ionomes.

Two lineages of Lemna aequinoctialis (Araceae, Lemnoideae) based on physiology, morphology, and phylogeny.

Lemna aequinoctialis Welw. is a widely spread species that has diverse physiological and molecular properties. Flower characteristics are important factors in deducing taxonomical status; however, owing to the rarity of flowering observations in Lemna, studying them has been a prolonged challenge. In this study, physiological and morphological analyses were conducted by inducing flowering, and molecular analysis was done based on the two chloroplast DNA loci (matK, atpF-atpH intergeneric spacer) of L. aequinoctialis sensu Landolt (1986) from 70 strains found in 70 localities in Japan, Korea, Thailand, and the US. In total, 752 flowering fronds from 13 strains were observed based on axenic conditions. Two different trends in flower organ development-protogyny and adichogamy-were detected in these strains. Their physiological traits were divided into two groups, showing different morphological features based on frond thickness, root cap, and anther sizes. Molecular analysis showed two lineages corresponding to two physiological groups. These were identified as L. aequinoctialis sensu Beppu et al. (1985) and L. aoukikusa Beppu et Murata based on the description of the nomenclature of L. aoukikusa. These were concluded as independent taxa and can be treated as different species. Furthermore, the distribution of L. aoukikusa is not only limited to Japan.

Distinct responses of frond and root to increasing nutrient availability in a floating clonal plant.

Current knowledge on responses of aquatic clonal plants to resource availability is largely based on studies manipulating limited resource levels, which may have failed to capture the "big picture" for aquatic clonal plants in response to resource availability. In a greenhouse experiment, we grew the floating clonal plant Spirodela polyrhiza under ten nutrient levels (i.e., 1/64×, 1/32×, 1/16×, 1/8×, 1/4×, 1/2×, 1×, 2×, 4× and 8×full-strength Hoagland solution) and examined their responses in terms of clonal growth, morphology and biomass allocations. The responses of total biomass and number of ramets to nutrient availability were unimodal. A similar pattern was found for frond mass, frond length and frond width, even though area per frond and specific frond area fluctuated greatly in response to nutrient availability. In contrast, the responses of root mass and root length to nutrient availability were U-shaped. Moreover, S. polyrhiza invested more to roots under lower nutrient concentrations. These results suggest that nutrient availability may have distinct influences on roots and fronds of the aquatic clonal plant S. polyrhiza, resulting in a great influence on the whole S. polyrhiza population.

Genome of the world's smallest flowering plant, Wolffia australiana, helps explain its specialized physiology and unique morphology.

Watermeal, Wolffia australiana, is the smallest known flowering monocot and is rich in protein. Despite its great potential as a biotech crop, basic research on Wolffia is in its infancy. Here, we generated the reference genome of a species of watermeal, W. australiana, and identified the genome-wide features that may contribute to its atypical anatomy and physiology, including the absence of roots, adaxial stomata development, and anaerobic life as a turion. In addition, we found evidence of extensive genome rearrangements that may underpin the specialized aquatic lifestyle of watermeal. Analysis of the gene inventory of this intriguing species helps explain the distinct characteristics of W. australiana and its unique evolutionary trajectory.

Plant evolution and environmental adaptation unveiled by long-read whole-genome sequencing of Spirodela.

Aquatic plants have to adapt to the environments distinct from where land plants grow. A critical aspect of adaptation is the dynamics of sequence repeats, not resolved in older sequencing platforms due to incomplete and fragmented genome assemblies from short reads. Therefore, we used PacBio long-read sequencing of the Spirodela polyrhiza genome, reaching a 44-fold increase of contiguity with an N50 (a median of contig lengths) of 831 kb and filling 95.4% of gaps left from the previous version. Reconstruction of repeat regions indicates that sequentially nested long terminal repeat (LTR) retrotranspositions occur early in monocot evolution, featured with both prokaryote-like gene-rich regions and eukaryotic repeat islands. Protein-coding genes are reduced to 18,708 gene models supported by 492,435 high-quality full-length PacBio complementary DNA (cDNA) sequences. Different from land plants, the primitive architecture of Spirodela's adventitious roots and lack of lateral roots and root hairs are consistent with dispensable functions of nutrient absorption. Disease-resistant genes encoding antimicrobial peptides and dirigent proteins are expanded by tandem duplications. Remarkably, disease-resistant genes are not only amplified, but also highly expressed, consistent with low levels of 24-nucleotide (nt) small interfering RNA (siRNA) that silence the immune system of land plants, thereby protecting Spirodela against a wide spectrum of pathogens and pests. The long-read sequence information not only sheds light on plant evolution and adaptation to the environment, but also facilitates applications in bioenergy and phytoremediation.

Toxic effects of Pb on Spirodela polyrhiza (L.): Subcellular distribution, chemical forms, morphological and physiological disorders.

The impact of lead (Pb) on Spirodela polyrhiza was studied to determine the subcellular distribution, chemical forms, and resulting morphophysiological modifications after treatments with 20 or 80 µM Pb(NO3)2 for 10 days. At the subcellular level, the Pb uptake by S. polyrhiza was mainly compartmentalized in the cell walls (70%), and the majority of Pb (approximately 70%) was extracted using 1 M NaCl and 2% acetic acid (HAc). Visual symptoms of phytotoxcity, surface roughness and closure of stomata, were observed in Pb-treated fronds. Electron-dense precipitates were present in cell walls, and changes to the ultrastructure were most noticeably exhibited in organelle shape, internal organization, and size of the plastoglobules of chloroplasts. Toxic concentrations of Pb induced oxidative stress in fronds, characterized by an accumulation of malondialdehyde (MDA) and decreased chlorophyll and unsaturated fatty acid contents. Pb exposure increased ABS/RC, TRo/RC, DIo/RC, Vj, and φDo (Fv/Fm), indicating that reaction centers were transformed to dissipation sinks, leading to a decrease in the efficiency of photosystem II, which was evident from the decreased values of Fv/Fo, Fv/Fm, ψEo, φEo, RC/ABS, and PIabs. These results indicated that decreased photosynthesis in Pb-treated fronds was partially ascribed to the lower pigment content, inhibition of electron transport, inactivation of the reaction centers, damage to the chloroplast ultrastructure, and stomatal closure. The physiological implications of subcellular distribution and chemical forms are discussed in relation to Pb accumulation and detoxification. However, Pb accumulation significantly impaired photosynthesis and membrane integrity in the fronds of S. polyrhiza.

Palyno-anatomical studies of monocot taxa and its taxonomic implications using light and scanning electron microscopy.

Palyno-anatomical study of monocots taxa using Light and Scanning Electron Microscopy (SEM) was first time conducted with a view to evaluating their taxonomic significance. Studied plants were collected from different eco-climatic zones of Pakistan ranges from tropical, sub-tropical, and moist habitats. The aim of this study is to use palyno-anatomical features for the correct identification, systematic comparison, and investigation to elucidate the taxonomic significance of these features, which are useful to taxonomists for identifying monocot taxa. A signification variation was observed in quantitative and qualitative characters by using the standard protocol of light microscopy (LM) and SEM. Epidermal cell length varied from maximum in Allium griffthianum (480 ± 35.9) µm at the adaxial surface to minimum in Canna indica (33.6 ± 8.53) µm on abaxial surface. Maximum exine thickness was observed in Canna indica (4.46) µm and minimum in Allium grifthianum (0.8) µm. Variation was observed in shape and exine ornamentation of the pollen, shape of the epidermal cell, number, size, and type of stomata, guard cell shape, and anticlinal wall pattern. Based on these palyno-anatomical features a taxonomic key was developed, which help in the discrimination of studied taxa. In conclusion, LM and SEM pollen and epidermal morphology is explanatory, significant, and can be of special interest for the plant taxonomist in the correct identification of monocots taxa.

Comparative profiling of microRNAs and their effects on abiotic stress in wild-type and dark green leaf color mutant plants of Anthurium andraeanum 'Sonate'.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including Anthurium andraeanum. To identify miRNAs and their target genes in A. andraeanum and study their responses to abiotic stresses, we conducted deep-sequencing of two small RNA (sRNA) libraries prepared from young leaves of wild-type (WT) and dark green (dg) leaf color mutant plants of A. andraeanum 'Sonate'. A total of 53 novel miRNAs were identified, 32 of which have been annotated to 18 miRNA families. 10 putative miRNAs were found to be differentially expressed in WT and dg, among which two miRNAs were significantly up-regulated and eight down-regulated in dg relative to WT. One differentially expressed miRNA, Aa-miR408, was dramatically up-regulated in dg. qRT-PCR analysis and heterologous expression of Aa-miR408 in Arabidopsis under different stress treatments suggest that Aa-miR408 is involved in abiotic stress responses in A. andraeanum. Our results provide a foundation for further dissecting the roles of miRNAs and their targets in regulating abiotic stress tolerance in A. andraeanum.

Near-infrared fluorescence imaging in the largely unexplored window of 900-1,000 nm.

Near-infrared (NIR) fluorescence imaging has relied on fluorophores that emit in the 700-900 nm NIR-Ia or 1,000-1,700 nm NIR-II window for generating deep-tissue images. Up until now, there have been few fluorophores developed for the 900-1,000 nm NIR-Ib window. This is largely because NIR-Ib light is thought to be strongly absorbed by water. Methods: Here we found that six heptamethine dyes had distinct emission peaks in both the NIR-Ia and NIR-Ib window. We tested the performance of these contrast agents by introducing them into the leaves of the common house plant Epipremnum aureum with early stage anthracnose leaf infections from Khaya senegalensis, as well as injecting them into the hind feet of nude mice and tails of tumour-bearing mice in vivo. Results: Heptamethine dyes yielded superior images of leaf venation, anthracnose infection locations, sentinel lymph nodes, brain tumours and subcutaneous tumours in the NIR-Ib window. We found that NIR-Ib images had markedly enhanced signal-to-background ratio because autofluorescence, scattering and light absorption by biological tissues and water were weaker at longer wavelengths. Conclusion: NIR-Ib fluorescence imaging was a powerful method for studying sentinel lymph nodes, tumours, leaf veins and early anthracnose infection locations in plant leaves. The findings challenge our current view of NIR fluorescence imaging and may have important implications for biomedical research and image-guided cancer surgery.

Morphological and structural characterization of the attachment system in aerial roots of Syngonium podophyllum.

MAIN CONCLUSION: The attachment of aerial roots of Syngonium podophyllum involves a multi-step process adjusted by multi-scale structures. Helical-crack root hairs are first found in the attachment system, representing specialized structures for surface anchorage. The morphological variability of attachment organs reflects diverse climbing strategies. One such anchoring mode in clinging-climbers involves the time-dependent interaction between roots and the support: By naturally occurring adhesive roots with root hairs, the plant can ascend on supports of any shape and size. As a typical root-climber, Syngonium podophyllum develops elongate aerial roots at nodes. Here, we studied its attachment behavior from the external morphology to the internal structure in detail. Through SEM and LM observation on several root-substrate interfaces, we suggested that the attachment of aerial roots was mediated by a multi-step process, in which root hairs played significant roles in releasing mucilaginous substance and securing the durable anchorage. We summarized all the types of shape changes of root hairs with particular focus on the abnormal transition from a tube to a helical-crack ribbon. We demonstrated our understanding with respect to the formation of the helical-crack root hairs, based on the structural evidence of cellulose microfibrils orientation on the cell wall lamellae. The helical-crack root hairs serving as energy-dissipating units retard the failure of adhesion under high winds and loads.

A multigenerational effect of parental age on offspring size but not fitness in common duckweed (Lemna minor).

Classic theories on the evolution of senescence make the simplifying assumption that all offspring are of equal quality, so that demographic senescence only manifests through declining rates of survival or fecundity. However, there is now evidence that, in addition to declining rates of survival and fecundity, many organisms are subject to age-related declines in the quality of offspring produced (i.e. parental age effects). Recent modelling approaches allow for the incorporation of parental age effects into classic demographic analyses, assuming that such effects are limited to a single generation. Does this 'single-generation' assumption hold? To find out, we conducted a laboratory study with the aquatic plant Lemna minor, a species for which parental age effects have been demonstrated previously. We compared the size and fitness of 423 laboratory-cultured plants (asexually derived ramets) representing various birth orders, and ancestral 'birth-order genealogies'. We found that offspring size and fitness both declined with increasing 'immediate' birth order (i.e. birth order with respect to the immediate parent), but only offspring size was affected by ancestral birth order. Thus, the assumption that parental age effects on offspring fitness are limited to a single generation does in fact hold for L. minor. This result will guide theorists aiming to refine and generalize modelling approaches that incorporate parental age effects into evolutionary theory on senescence.

Flowering mechanisms, pollination strategies and floral scent analyses of syntopically co-flowering Homalomena spp. (Araceae) on Borneo.

In this study, the flowering mechanisms and pollination strategies of seven species of the highly diverse genus Homalomena (Araceae) were investigated in native populations of West Sarawak, Borneo. The floral scent compositions were also recorded for six of these species. The selected taxa belong to three out of four complexes of the section Cyrtocladon (Hanneae, Giamensis and Borneensis). The species belonging to the Hanneae complex exhibited longer anthesis (53-62 h) than those of the Giamensis and Borneensis complexes (ca. 30 h). Species belonging to the Hanneae complex underwent two floral scent emission events in consecutive days, during the pistillate and staminate phases of anthesis. In species belonging to the Giamensis and Borneensis complexes, floral scent emission was only evident to the human nose during the pistillate phase. A total of 33 volatile organic compounds (VOCs) were detected in floral scent analyses of species belonging to the Hanneae complex, whereas 26 VOCs were found in samples of those belonging to the Giamensis complex. The floral scent blends contained uncommon compounds in high concentration, which could ensure pollinator discrimination. Our observations indicate that scarab beetles (Parastasia gestroi and P. nigripennis; Scarabaeidae, Rutelinae) are the pollinators of the investigated species of Homalomena, with Chaloenus schawalleri (Chrysomelidae, Galeuricinae) acting as a secondary pollinator. The pollinators utilise the inflorescence for food, mating opportunities and safe mating arena as rewards. Flower-breeding flies (Colocasiomyia nigricauda and C. aff. heterodonta; Diptera, Drosophilidae) and terrestrial hydrophilid beetles (Cycreon sp.; Coleoptera, Hydrophilidae) were also frequently recovered from inflorescences belonging to all studied species (except H. velutipedunculata), but they probably do not act as efficient pollinators. Future studies should investigate the post-mating isolating barriers among syntopically co-flowering Homalomena sharing the same visiting insects.

Floral diversity and pollination strategies of three rheophytic Schismatoglottideae (Araceae).

Homoplastic evolution of 'unique' morphological characteristics in the Schismatoglottideae - many previously used to define genera - prompted this study to compare morphology and function in connection with pollination biology for Aridarum nicolsonii, Phymatarum borneense and Schottarum sarikeense. Aridarum nicolsonii and P. borneense extrude pollen through a pair of horned thecae while S. sarikeense sheds pollen through a pair of pores on the thecae. Floral traits of spathe constriction, presence and movement of sterile structures on the spadix, the comparable role of horned thecae and thecae pores, the presence of stamen-associated calcium oxalate packages, and the timing of odour emission are discussed in the context of their roles in pollinator management. Pollinators for all investigated species were determined to be species of Colocasiomyia (Diptera: Drosophilidae).

Phytotoxicity of cobalt ions on the duckweed Lemna minor - Morphology, ion uptake, and starch accumulation.

Cobalt (Co2+) inhibits vegetative growth of Lemna minor gradually from 1 µM to 100 µM. Fronds accumulated up to 21 mg Co2+ g(-1) dry weight at 10 µM external Co2+ indicating hyperaccumulation. Interestingly, accumulation of Co2+ did not decrease the iron (Fe) content in fronds, highlighting L. minor as a suitable system for studying effects of Co2+ undisturbed by Fe deficiency symptoms unlike most other plants. Digital image analysis revealed the size distribution of fronds after Co2+ treatment and also a reduction in pigmentation of newly formed daughter fronds unlike the mother fronds during the 7-day treatment. Neither chlorophyll nor photosystem II fluorescence changed significantly during the initial 4d, indicating effective photosynthesis. During the later phase of the 7-day treatment, however, chlorophyll content and photosynthetic efficiency decreased in the Co2+-treated daughter fronds, indicating that Co2+ inhibits the biosynthesis of chlorophyll rather than leading to the destruction of pre-existing pigment molecules. In addition, during the first 4d of Co2+ treatment starch accumulated in the fronds and led to the transition of chloroplasts to chloro-amyloplasts and amylo-chloroplasts, while starch levels strongly decreased thereafter.

Specific turion yields of different clones of Spirodela polyrhiza depend on external phosphate thresholds.

Turions play an important role in the survival strategy of the duckweed Spirodela polyrhiza. Therefore, factors influencing the formation of these survival organs were studied. Phosphate deficiency is the main natural factor inducing turion formation and the specific turion yield (SY), i.e. the number of turions formed per frond, varied widely for five different clones from different climate zones. The concentrations of phosphate and nitrate in the nutrient media were investigated at the onset of turion formation, with SY ranging from 0.22 to 5.9. Tissue P and N content was also investigated in vegetative fronds at the onset of turion formation and in newly formed turions. The clones were selected to test possible correlations between SY and threshold nutrient concentration for turion formation. Only one correlation, between SY and external phosphate concentration, was significant: clones with high SY started turion formation at higher external phosphate concentrations. Turion formation is thus mainly induced by the external phosphate concentration, below a defined, clone-dependent threshold. We propose the following mechanism: a switch of the developmental programme of frond primordia from vegetative frond to turion formation at a higher phosphate threshold saves more phosphate for turion formation instead of using it for a further vegetative growth. However, the period of growth preceding turion formation does not depend on this threshold value, but rather on the growth rate of the vegetative fronds, which actually produce the phosphate shortage by taking it up from the surrounding medium.

Chromatin organisation in duckweed interphase nuclei in relation to the nuclear DNA content.

The accessibility of DNA during fundamental processes, such as transcription, replication and DNA repair, is tightly modulated through a dynamic chromatin structure. Differences in large-scale chromatin structure at the microscopic level can be observed as euchromatic and heterochromatic domains in interphase nuclei. Here, key epigenetic marks, including histone H3 methylation and 5-methylcytosine (5-mC) as a DNA modification, were studied cytologically to describe the chromatin organisation of representative species of the five duckweed genera in the context of their nuclear DNA content, which ranged from 158 to 1881 Mbp. All studied duckweeds, including Spirodela polyrhiza with a genome size and repeat proportion similar to that of Arabidopsis thaliana, showed dispersed distribution of heterochromatin signatures (5mC, H3K9me2 and H3K27me1). This immunolabelling pattern resembles that of early developmental stages of Arabidopsis nuclei, with less pronounced heterochromatin chromocenters and heterochromatic marks weakly dispersed throughout the nucleus.

Growing duckweed for biofuel production: a review.

Duckweed can be utilised to produce ethanol, butanol and biogas, which are promising alternative energy sources to minimise dependence on limited crude oil and natural gas. The advantages of this aquatic plant include high rate of nutrient (nitrogen and phosphorus) uptake, high biomass yield and great potential as an alternative feedstock for the production of fuel ethanol, butanol and biogas. The objective of this article is to review the published research on growing duckweed for the production of the biofuels, especially starch enrichment in duckweed plants. There are mainly two processes affecting the accumulation of starch in duckweed biomass: photosynthesis for starch generation and metabolism-related starch consumption. The cost of stimulating photosynthesis is relatively high based on current technologies. Considerable research efforts have been made to inhibit starch degradation. Future research need in this area includes duckweed selection, optimisation of duckweed biomass production, enhancement of starch accumulation in duckweeds and use of duckweeds for production of various biofuels.

Status of duckweed genomics and transcriptomics.

Duckweeds belong to the smallest flowering plants that undergo fast vegetative growth in an aquatic environment. They are commonly used in wastewater treatment and animal feed. Whereas duckweeds have been studied at the biochemical level, their reduced morphology and wide environmental adaption had not been subjected to molecular analysis until recently. Here, we review the progress that has been made in using a DNA barcode system and the sequences of chloroplast and mitochondrial genomes to identify duckweed species at the species or population level. We also review analysis of the nuclear genome sequence of Spirodela that provides new insights into fundamental biological questions. Indeed, reduced gene families and missing genes are consistent with its compact morphogenesis, aquatic floating and suppression of juvenile-to-adult transition. Furthermore, deep RNA sequencing of Spirodela at the onset of dormancy and Landoltia in exposure of nutrient deficiency illustrate the molecular network for environmental adaption and stress response, constituting major progress towards a post-genome sequencing phase, where further functional genomic details can be explored. Rapid advances in sequencing technologies could continue to promote a proliferation of genome sequences for additional ecotypes as well as for other duckweed species.

Molecular characterization of Anthurium genotypes by using DNA fingerprinting and SPAR markers.

We characterized single primer amplification reaction (SPAR) molecular markers from 20 genotypes of Anthurium andraeanum Lind., including 3 from commercial varieties and 17 from 2 communities in the State of Espírito Santo, Brazil. Twenty-four SPAR, consisting of 7 random amplified polymorphic DNA and 17 inter-simple sequence repeat markers were used to estimate the genetic diversity of 20 Anthurium accessions. The set of SPAR markers generated 288 bands and showed an average polymorphism percentage of 93.39%, ranging from 71.43 to 100%. The polymorphism information content (PIC) of the random amplified polymorphic DNA primers averaged 0.364 and ranged from 0.258 to 0.490. Primer OPF 06 showed the lowest PIC, while OPAM 14 was the highest. The average PIC of the inter-simple sequence repeat primers was 0.299, with values ranging from 0.196 to 0.401. Primer UBC 845 had the lowest PIC (0.196), while primer UCB 810 had the highest (0.401). By using the complement of Jaccard's similarity index and unweighted pair group method with arithmetic mean clustering, 5 clusters were formed with a cophenetic correlation coefficient of 0.8093, indicating an acceptable clustering consistency. However, no genotype clustering patterns agreed with the morphological data. The Anthurium genotypes investigated in this study are a germplasm source for conservational research and may be used in improvement programs for this species.

Adaptations for insect-trapping in brood-site pollinated Colocasia (Araceae).

The Araceae include both taxa with rewarding and deceptive trap pollination systems. Here we report on a genus in which rewarding and imprisonment of the pollinators co-occur. We studied the pollination of four species of Colocasia in Southwest China and investigated the morpho-anatomical adaptations of the spathe related to the attraction and capture of pollinators. All four species were pollinated by drosophilid flies of the genus Colocasiomyia. The flies are temporally arrested within the inflorescence and departure is only possible after pollen release. Trapping of the flies is accomplished by the closure of the spathe during anthesis. Moreover, in two species the spathe is covered with papillate epidermal cells known to form slippery surfaces in deceptive traps of Araceae. However, in Colocasia the papillae proved not slippery for the flies. The morpho-anatomical properties of the spathe epidermis indicate that it is an elaborate osmophore and serves for the emission of odours only. Despite its similarity to deceptive traps of other aroids, Colocasia and Colocasiomyia have a close symbiotic relationship, as the attracted flies use the inflorescence as a site for mating and breeding. The trap mechanism has presumably evolved independently in Colocasia and is supposed to facilitate more efficient pollen export.