Late Devonian (Frasnian) phyllopod and phyllocarid crustacean shields from Belgium reinterpreted as ammonoid anaptychi

The taxonomic affinities of fossils from the Frasnian succession of Belgium previously described as phyllopod and phyllocarid crustacean shields are discussed. The rediscovery of the holotype of Ellipsocaris dewalquei, the type species of the genus Ellipsocaris Woodward in Dewalque, 1882, allows to end the discussion on the taxonomic assignation of the genus Ellipsocaris. It is removed from the phyllopod crustaceans as interpreted originally and considered here as an ammonoid anaptychus. Furthermore, it is considered to be a junior synonym of the genus Sidetes Giebel, 1847. Similarly, Van Straelen’s (1933) lower to middle Frasnian record Spathiocaris chagrinensis Ruedemann, 1916, is also an ammonoid anaptychus. Although ammonoids can be relatively frequent in some Frasnian horizons of Belgium, anaptychi remain particularly scarce and the attribution to the present material to peculiar ammonoid species is not possible.     

Computational molecular species delimitation and taxonomic revision of the gecko genus <Emphasis Type="Italic">Ebenavia</Emphasis> Boettger, 1878

Cryptic species have been detected in many groups of organisms and must be assumed to make up a significant portion of global biodiversity. We study geckos of the Ebenavia inunguis complex from Madagascar and surrounding islands and use species delimitation algorithms (GMYC, BOLD, BPP), COI barcode divergence, diagnostic codon indels in the nuclear marker PRLR, diagnostic categorical morphological characters, and significant differences in continuous morphological characters for its taxonomic revision. BPP yielded ≥?10 operational taxonomic units, whereas GMYC (≥?27) and BOLD (26) suggested substantial oversplitting. In consequnce, we resurrect Ebenavia boettgeri Boulenger 1885 and describe Ebenavia tuelinae sp. nov., Ebenavia safari sp. nov., and Ebenavia robusta sp. nov., increasing the number of recognised species in Ebenavia from two to six. Further lineages of Ebenavia retrieved by BPP may warrant species or subspecies status, but further taxonomic conclusions are postponed until more data become available. Finally, we present an identification key to the genus Ebenavia, provide an updated distribution map, and discuss the diagnostic values of computational species delimitation as well as morphological and molecular diagnostic characters.     

The onion fly modulates the adult eclosion time in response to amplitude of temperature cycle

To confirm whether the amplitude of diel temperature cycles causes a phase shift of adult eclosion rhythm of the onion fly, Delia antiqua, the peak time (Ø _E) of adult eclosion was determined under various thermoperiods with a fixed temperature either in the warm or cool phase and temperature differences ranging from 1°C to 4°C between the two phases. Irrespective of the temperature level during the warm or cool phase, Ø _E occurred earlier with decreasing amplitude of the temperature cycle. The results strongly support the previous conclusion of Tanaka and Watari (Naturwissenschaften 90:76–79, 2003) that D. antiqua responds to the amplitude of temperature cycle as a cue for the circadian adult eclosion timing. The phase advance was larger in thermoperiods with a fixed warm-phase temperature than in those with a fixed cool-phase temperature. This might be ascribed to the interaction between the amplitude and level of temperature in the thermoperiodic regimes.     

Marginal costs of abating greenhouse gases in the global ruminant livestock sector

Livestock [inclusive of ruminant species, namely cattle (Bos Taurus and Bos indicus), sheep (Ovis aries), goats (Capra hircus), and buffaloes (Bubalus bubalis), and non-ruminant species, namely pigs (Sus scrofa domesticus) and chickens (Gallus domesticus)] are both affected by climate change and contribute as much as 14.5 % of global anthropogenic greenhouse gas (GHG) emissions, most of which is from ruminant animals (Gerber et al. 2013). This study aims to estimate the marginal costs of reducing GHG emissions for a selection of practices in the ruminant livestock sector (inclusive of the major ruminant species—cattle, sheep, and goats) globally. It advances on previous assessments by calculating marginal costs rather than commonly reported average costs of abatement and can thus provide insights about abatement responses at different carbon prices. We selected the most promising abatement options based on their effectiveness and feasibility. Improved grazing management and legume sowing are the main practices assessed in grazing systems. The urea (CO(NH₂)₂) treatment of crop straws is the main practice applied in mixed crop–livestock systems, while the feeding of dietary lipids and nitrates are confined to more intensive production systems. These practices were estimated to reduce emissions by up to 379 metric megatons of carbon dioxide (CO₂) equivalent emissions per year (MtCO₂-eq yr^?1). Two thirds of this reduction was estimated to be possible at a carbon price of 20 US dollars per metric ton of CO₂ equivalent emissions ($20 tCO₂-eq^?1). This study also provides strategic guidance as to where abatement efforts could be most cost effectively targeted. For example, improved grazing management was particularly cost effective in Latin America and Sub-Saharan Africa, while legume sowing appeared to work best in Western Europe and Latin America.     

Olfaction in a viscous environment: the “color” of sexual smells in <Emphasis Type="Italic">Temora longicornis</Emphasis>

We investigate chemical aspects of mating in the marine copepod Temora longicornis (Copepoda, Calanoidea). Our emphasis is the female pheromone signaling in form of well-defined trails for males to follow, observed in Doall et al. (Phil Trans R Soc Lond B 353:681–689, 1998). The viscous environment and the properties of the odorants play important roles as the spread of the pheromone trail limits the time during which it is useful for tracing. A key observation from our earlier work is the ability of a searching male to detect the direction of the female and to correct its swimming direction if necessary. We propose a simple mathematical model for the spread of a pheromone from a moving source and carry out numerical simulations of two possible detection mechanisms. We find that a searching agent that is capable to detect a ratio outperforms a searcher that depends on the gradient of a single compound. This suggests that copepod sex pheromones consist of blends of chemical compounds, and that a ratio detection mechanism similar to that in airborne insects is at work.     

Influence of tree species composition,thinning intensity and climate change on carbon sequestration in Mediterranean mountain forests: a case study using the CO2Fix model

Prediction of future forest carbon (C) stocks as influenced by forest management and climate is a crucial issue in the search for strategies to mitigate and adapt to global change. It is hard to quantify the long-term effect of specific forest practices on C stocks due to the high number of processes affected by forest management. This work aims to quantify how forest management impacts C stocks in Mediterranean mountain forests based on 25 combinations of site index, tree species composition and thinning intensity in three different climate scenarios using the CO2Fix v.3.2 model Masera et al. (Ecol Modell 164:177–199, 2003). The study area is an ecotonal zone located in Central Spain, and the tree species are Scots pine (Pinus sylvestris L.) and Pyrenean oak (Quercus pyrenaica Willd.). Our results show a strong effect of tree species composition and a negligible effect of thinning intensity. Mixed stands have the highest total stand C stocks: 100 % and 15 % more than pure oak and pine stands respectively, and are here suggested as a feasible and effective mitigation option. Climate change induced a net C loss compared to control scenarios, when reduction in tree growth is taken into account. Mixed stands showed the lowest reduction in forest C stocks due to climate change, indicating that mixed stands are also a valid adaptation strategy. Thus converting from pure to mixed forests would enhance C sequestration under both current and future climate conditions.     

Three-year lifecycle,large body,and very high threshold temperature in the cricket <Emphasis Type="Italic">Gryllus argenteus</Emphasis> for special adaptation to desiccation cycle in Malawi

In temperate climates, the initiation and termination of diapause synchronize the stress-tolerant stage with the stressful season and reproduction with the non-stressful season in many insects. Synchronization is often regulated by photoperiodism.Voltinism and the ultimate size of adults are also important determinants for their lifecycle, and different diapause stages and voltinism patterns are known in crickets.Here, we investigated the life history of the African cricket Gryllus argenteus from Malawi, which is a typical arid tropical highland. The climate is characterized by alternating arid and wet seasons, each of which lasts for half a year, and where the available heat mass is much less than lowlands at the same latitude. We first measured the nymphal duration at each rearing temperature and calculated the lower developmental threshold (t ₀) to be 20.19 °C based on Ikemoto and Takai (2000) and 19.38 °C based on a conventional line-fitting method. These values are very high relative to many other insects. The local temperature in winter does not fall below 15 °C, but this is much higher than the lethal limit. This suggested that critical stress in this locality was not coldness but low precipitation in winter. We estimated, based both on local temperature change and the Ikemoto and Takai’s t ₀, that G. argenteus required 3 years to complete its lifecycle unlike wet lowland species, where univoltinism or multi-voltinism are commonplace. Photoperiodism was observed in this species, but due to a lag between annual cycles in photoperiod, temperature, and humidity, photoperiodism alone cannot atune their lifecycle with local conditions.Synchronization in this species was achieved by three different adaptations: photoperiodism, high t ₀, and large body size, which give it a long lifecycle. Although the species cannot achieve a univoltine lifecycle because of its high t₀ value, it can escape from dry season by entering diapause at moderate temperatures, probably thereby achieving adaptive synchrony of lifecycle with both favorable and unfavorable seasons. A comparison between a conventional photothermogram and a newly formulated photohydrogram or photohygrogram demonstrates that even though sufficient heat is available, scarcity of water and thus scarcity of foliage should force the cricket to maintain diapause at intermediate temperature. The results suggested that high t ₀, large body size, and multi-ennial lifecycle mutually affect each other and formulate a unique adaptation under such an extreme environment.     

The power and pain of market-based carbon policies: a global application to greenhouse gases from ruminant livestock production

The objectives of this research are to assess the greenhouse gas mitigation potential of carbon policies applied to the ruminant livestock sector [inclusive of the major ruminant species—cattle (Bos Taurus and Bos indicus), sheep (Ovis aries), and goats (Capra hircus)]—with particular emphasis on understanding the adjustment challenges posed by such policies. We show that market-based mitigation policies can greatly amplify the mitigation potential identified in marginal abatement cost studies by harnessing powerful market forces such as product substitution and trade. We estimate that a carbon tax of US$20 per metric ton of carbon dioxide (CO₂) equivalent emissions could mitigate 626 metric megatons of CO₂ equivalent ruminant emissions per year (MtCO₂-eq year^?1). This policy would also incentivize a restructuring of cattle production, increasing the share of cattle meat coming from the multiproduct dairy sector compared to more emission intensive, single purpose beef sector. The mitigation potential from this simple policy represents an upper bound because it causes ruminant-based food production to fall and is therefore likely to be politically unpopular. In the spirit of the Paris Agreement (UNFCCC 2015), which expresses the ambition of reducing agricultural emissions while protecting food production, we assess a carbon policy that applies both a carbon tax and a subsidy to producers to manage the tradeoff between food production and mitigation. The policy maintains ruminant production and consumption levels in all regions, but for a much lower global emission reduction of 185 MtCO₂-eq year^?1. This research provides policymakers with a quantitative basis for designing policies that attempt to trade off mitigation effectiveness with producer and consumer welfare.     

Subfossil markers of climate change during the Roman Warm Period of the late Holocene

Abundant bog oak trunks occur in alluvial deposits of the Raba River in the village of Targowisko (southern Poland). Several of them contain galleries of the great capricorn beetle (Cerambyx cerdo L.). A well-preserved subfossil larva and pupa, as well as adults of this species, are concealed in some of the galleries. These galleries co-occur with boring galleries of other insects such as ship-timber beetles (Lymexylidae) and metallic wood borers (Buprestidae). A dry larva of a stag beetle (Lucanidae) and a mite (Acari) have been found in the C. cerdo galleries. Selected samples of the trunks and a sample of the C. cerdo larva were dated, using radiocarbon and dendrochronological methods, to the period from 45 bc to ad 554; one sample was dated to the period from 799 to 700 bc. Accumulation of the channel alluvia containing the bog oak trunks is synchronous with the Roman Warm Period (late antiquity/Early Mediaeval times). The most recent part of this period correlates with massive accumulations of fallen oak trunks noted from various river valleys in the Carpathian region and dated to ad 450–570. The results indicate that C. cerdo was more abundant within the study area during the Roman Warm Period than it is today.     

Mathematical model for the microbial metabolism of glucose induced enhanced biological phosphorus removal in an anaerobic/aerobic sequential batch reactor

Glucose plays a significant role in enhanced biological phosphorus removal (EBPR). Previous experimental studies have shown that in addition to acetate, glucose can induce and maintain a successful EBPR performance under certain operating procedures. Mathematical modeling of the EBPR metabolism is an important consideration that is necessary to produce a deeper insight into this process. In this study, four anaerobic reactions plus four aerobic reactions with fourteen kinetic constants are structured to describe the dynamic behavior of the key metabolic components in the glucose induced EBPR system. The development of the stoichiometric coefficients for the reactions is based on fundamental biochemical principles. The model describes the dynamics of the important storage compounds, which are considered separately from the active biomass. Sequential batch experiments were performed to find the optimum model parameters using sludges exhibiting stable EBPR from glucose fed system. The maximum specific substrate conversion rate in the glucose model is composed of two factors: the maximum specific rates of glucose direct conversion to PHV (q _gv ^max ) and glucose conversion to glycogen q _gl ^max , which are 14.1 and 699.7 C-mmol/C-mmol·h, respectively. The maximum specific rate of polyphosphate synthesis k _pp ^max is 0.2 P-mmol/C-mmol·h. The model with the best-fit parameters satisfactorily simulates the dynamic behavior of the key components during both anaerobic and aerobic conditions.     

Using taphonomy to infer differences in soft tissues between taxa: an example using basal and derived forms of Solnhofen pterosaurs

In fossilised vertebrates, the presence of soft tissues is the most obvious way to determine aspects of anatomy and functional morphology; however, occurrences are rare and other lines of evidence must be sought to indicate its extent and strength. For example, pterosaurs possessed a large wing membrane that enabled powered flight but other tissues are not widely preserved. A semi-quantitative analysis comparing skeletal articulation and completeness of the pterodactyloid Pterodactylus and non-pterodactyloid pterosaur Rhamphorhynchus from Solnhofen-type deposits implies there were anatomical differences between soft-tissue structure and attachments articulating skeletal joints of each. Typically, skeletons of Pterodactylus disarticulate to a greater extent than those of Rhamphorhynchus, which in turn suggests decay progressed to more advanced states in the former. However, this generalisation masks a mosaic of differences between different body parts, for example Rhamphorhynchus tends to lose the wings as complete units but retains a complete and still articulated tail in a greater number of specimens than Pterodactylus.     

Microanatomy and life history in <Emphasis Type="Italic">Palaeopleurosaurus</Emphasis> (Rhynchocephalia: Pleurosauridae) from the Early Jurassic of Germany

The tuatara (Sphenodon punctatus) from New Zealand is often—erroneously—identified as a ‘living fossil’, although it is the lone survivor of a large, successful radiation of Rhynchocephalia, sister taxon to squamates (lizards and snakes), that thrived through the Mesozoic and Cenozoic and experienced an intricate evolution of life histories and feeding habits. Within Rhynchocephalia, only Pleurosauridae are thought to be marine and piscivorous. Here, we present bone histological data of the Jurassic pleurosaurid Palaeopleurosaurus, showing osteosclerosis (i.e. bone mass increase) in its gastralia, and some osteosclerosis in its rib but no increase in bone mass in the femur, supporting a gradual skeletal specialization for an aquatic way of life. Similar to Sphenodon, the bone tissue deposited in Palaeopleurosaurus is lamellar zonal bone. The femoral growth pattern in Palaeopleurosaurus differs from that of terrestrial Sphenodon in a more irregular spacing of growth marks and deposition of non-annual (i.e. non-continuous) rest lines, indicating strong dependency on exogenous factors. The annual growth mark count in adult but not yet fully grown Palaeopleurosaurus is much lower when compared to adult individuals of Sphenodon, which could indicate a lower lifespan for Palaeopleurosaurus. Whereas the gastral ribs of Palaeopleurosaurus and Sphenodon are similar in composition, the ribs of Sphenodon differ profoundly in being separated into a proximal tubular rib part with a thick cortex, and an elliptical, flared ventral part characterised by extremely thin cortical bone. The latter argues against a previously inferred protective function of the ventral rib parts for the vulnerable viscera in Sphenodon.     

Extreme longevity in a deep-sea vestimentiferan tubeworm and its implications for the evolution of life history strategies

The deep sea is home to many species that have longer life spans than their shallow-water counterparts. This trend is primarily related to the decline in metabolic rates with temperature as depth increases. However, at bathyal depths, the cold-seep vestimentiferan tubeworm species Lamellibrachia luymesi and Seepiophila jonesi reach extremely old ages beyond what is predicted by the simple scaling of life span with body size and temperature. Here, we use individual-based models based on in situ growth rates to show that another species of cold-seep tubeworm found in the Gulf of Mexico, Escarpia laminata, also has an extraordinarily long life span, regularly achieving ages of 100–200 years with some individuals older than 300 years. The distribution of results from individual simulations as well as whole population simulations involving mortality and recruitment rates support these age estimates. The low 0.67% mortality rate measurements from collected populations of E. laminata are similar to mortality rates in L. luymesi and S. jonesi and play a role in evolution of the long life span of cold-seep tubeworms. These results support longevity theory, which states that in the absence of extrinsic mortality threats, natural selection will select for individuals that senesce slower and reproduce continually into their old age.     

Vegaviidae,a new clade of southern diving birds that survived the K/T boundary

The fossil record of Late Cretaceous–Paleogene modern birds in the Southern Hemisphere includes the Maastrichtian Neogaeornis wetzeli from Chile, Polarornis gregorii and Vegavis iaai from Antarctica, and Australornis lovei from the Paleogene of New Zealand. The recent finding of a new and nearly complete Vegavis skeleton constitutes the most informative source for anatomical comparisons among Australornis, Polarornis, and Vegavis. The present contribution includes, for the first time, Vegavis, Polarornis, and Australornis in a comprehensive phylogenetic analysis. This analysis resulted in the recognition of these taxa as a clade of basal Anseriformes that we call Vegaviidae. Vegaviids share a combination of characters related to diving adaptations, including compact and thickened cortex of hindlimb bones, femur with anteroposteriorly compressed and bowed shaft, deep and wide popliteal fossa delimited by a medial ridge, tibiotarsus showing notably proximally expanded cnemial crests, expanded fibular crest, anteroposterior compression of the tibial shaft, and a tarsometatarsus with a strong transverse compression of the shaft. Isolated bones coming from the Cretaceous and Paleogene of South America, Antarctica, and New Zealand are also referred to here to Vegaviidae and support the view that these basal anseriforms were abundant and diverse at high southern latitudes. Moreover, vegaviids represent the first avian lineage to have definitely crossed the K–Pg boundary, supporting the idea that some avian clades were not affected by the end Mesozoic mass extinction event, countering previous interpretations. Recognition of Vegaviidae indicates that modern birds were diversified in southern continents by the Cretaceous and reinforces the hypothesis indicating the important role of Gondwana for the evolutionary history of Anseriformes and Neornithes as a whole.     

Evolution through cold and deep waters: the molecular phylogeny of the Lithodidae (Crustacea: Decapoda)

The objectives of this work are to use gene sequence data to assess the hypothesis that the Lithodinae arose from ancestors with uncalcified abdomens in shallow waters of the North-East Pacific, investigate the monophyly and interrelationships of genera within the Lithodinae and to estimate the scale and minimum number of biogeographic transitions from the shallow environment to the deep sea and vice versa. To do this, phylogenetic analysis from three mitochondrial and three nuclear markers was conducted using minimum evolution, maximum likelihood and Bayesian methods. The Lithodinae as defined to include North Pacific genus Cryptolithodes may be paraphyletic, with the Hapalogastrinae and Cryptolithodes as sister taxa. This implies that the soft-bodied abdomen of the Hapalogastrinae might not be plesiomorphic for the Lithodidae. Paralomis, Lopholithodes, Phyllolithodes, Lithodes and Neolithodes share a common ancestor, from which the North Pacific Hapalogastrinae did not descend. Lithodid ancestors are likely to have had a north Pacific, shallow water distribution and to have had planktotrophic larvae. North Pacific genus Paralithodes is paraphyletic; P. brevipes is the most basal member of the genus (as sampled) while P. camtschaticus and P. platypus are more closely related to the genera Lithodes and Neolithodes. Genera Lithodes, Neolithodes and Paralomis (as sampled) are monophyletic if Glyptolithodes is included within Paralomis. Lopholithodes is closely related to, but not included within, the Paralomis genus. Paralomis is divided into at least two major lineages: one containing South Atlantic, West African, and Indian Ocean species, and the other containing Pacific and South American species. Several species of Paralomis do not resolve consistently with any other groups sampled, implying a complex and possibly rapid global evolution early in the history of the genus. Relationships within the Lithodes genus vary between analytical methods, suggesting that conclusions may not be stable. Consistently, however, Indian Ocean and Pacific forms—L. murrayi, L. longispina and L. nintokuae form a group separated from Atlantic species such as L. santolla, L. confundens, L. maja and L. ferox.     

Antifungal activity of the pygidial gland secretion of <Emphasis Type="Italic">Laemostenus punctatus</Emphasis> (Coleoptera: Carabidae) against cave-dwelling micromycetes

The antifungal potential of the pygidial gland secretion of the troglophilic ground beetle Laemostenus punctatus from a cave in Southeastern Serbia against cave-dwelling micromycetes, isolated from the same habitat, has been investigated. Eleven collected samples were analyzed and 32 isolates of cave-dwelling fungi were documented. A total of 14 fungal species were identified as members of the genera Aspergillus, Penicillium, Alternaria, Cladosporium, Rhizopus, Trichoderma, Arthrinium, Aureobasidium, Epicoccum, Talaromyces, and Fusarium. Five isolates were selected for testing the antifungal activity of the pygidial gland secretion: Talaromyces duclauxi, Aspergillus brunneouniseriatus, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. The microdilution method has been applied to detect minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs). The most sensitive isolate was Penicillium sp., while the other isolates demonstrated a high level of resistance to the tested agent. L. punctatus has developed a special mechanism of producing specific compounds that act synergistically within the secretion mixture, which are responsible for the antifungal action against pathogens from the cave. The results open opportunities for further research in the field of ground beetle defense against pathogens, which could have an important application in human medicine, in addition to the environmental impact, primarily.     

Testing the influence of gravity on flower symmetry in five <Emphasis Type="Italic">Saxifraga</Emphasis> species

Flower symmetry is considered a species-specific trait and is categorized in asymmetry, actinomorphic symmetry, bisymmetry and zygomorphic symmetry. Here we report on the intra-individual variation of flower symmetry in the genus Saxifraga and the influence of light, gravity and intrinsic factors on the development of flower symmetry. We tested five species—Saxifraga cuneifolia, Saxifraga imparilis, Saxifraga rotundifolia, Saxifraga stolonifera and Saxifraga umbrosa—concerning six flower parameters—angles between petals, petal length, petal pigmentation, angular position of carpels, movement of stamens and (only for S. imparilis and S. stolonifera) the length of the two lower elongated petals in regard to their position towards the stem. Specimens of all species were tested on a vertical clinostat as a gravity compensator, on a horizontal clinostat as a light incidence compensator and on a stationary control. The results show that the angle of incident light has no apparent impact on flower symmetry, whereas gravity affects the angular position of petals in S. cuneifolia and S. umbrosa and the petal colouration in S. rotundifolia. In S. cuneifolia and S. umbrosa, the absence of directional gravity resulted in the development of actinomorphic flowers, whereas the corresponding control flowers were zygomorphic. The development of flowers in S. rotundifolia was not altered by this treatment. The length of the two elongated petals in S. stolonifera and S. imparilis was not affected by gravity, but rather was determined by position of the flower within the inflorescence and resulted in asymmetrical flowers.     

Combining climatic and soil properties better predicts covers of Brazilian biomes

Several techniques have been used to model the area covered by biomes or species. However, most models allow little freedom of choice of response variables and are conditioned to the use of climate predictors. This major restriction of the models has generated distributions of low accuracy or inconsistent with the actual cover. Our objective was to characterize the environmental space of the most representative biomes of Brazil and predict their cover, using climate and soil-related predictors. As sample units, we used 500 cells of 100 km² for ten biomes, derived from the official vegetation map of Brazil (IBGE 2004). With a total of 38 (climatic and soil-related) predictors, an a priori model was run with the random forest classifier. Each biome was calibrated with 75% of the samples. The final model was based on four climate and six soil-related predictors, the most important variables for the a priori model, without collinearity. The model reached a kappa value of 0.82, generating a highly consistent prediction with the actual cover of the country. We showed here that the richness of biomes should not be underestimated, and that in spite of the complex relationship, highly accurate modeling based on climatic and soil-related predictors is possible. These predictors are complementary, for covering different parts of the multidimensional niche. Thus, a single biome can cover a wide range of climatic space, versus a narrow range of soil types, so that its prediction is best adjusted by soil-related variables, or vice versa.