A Comparison of the Composition of Wax Ester Molecular Species of Different Coral Groups (Subclasses Hexacorallia and Octocorallia)

Wax esters, which are esters of fatty alcohols and fatty acids (FAs), are one of the main classes of reserve lipids in all coral species. The chemical structures and the content of wax ester molecular species were determined for the first time in nine coral species from three taxonomic groups: symbiotic reef-building corals, (Hexacorallia subclasses), symbiotic soft coral alcyonarians, and asymbiotic soft coral gorgonians (Octocorallia subclasses) collected in the South China Sea (Vietnam). Our comparison of these groups showed that the absence of symbiotic microalgae (zooxanthellae) and the exoskeleton affects the profile of molecular species of wax esters considerably. The main components of wax esters of all corals were cetyl palmitate (16:0-16:0) and other saturated wax esters containing 30, 34, and 36 carbon atoms. The content of unsaturated molecular species 6:0–16:1, 16:0–18:1, and 16:0–20:1 in wax esters of symbiotic soft corals (alcyonarians) was greater than that in wax esters of reef-building corals. In contrast to symbiotic coral species, wax esters of asymbiotic soft corals, namely azooxanthellate gorgonians, contained a considerable amount of long-chain molecular species (C₃₇-C₄₁) with an odd number of carbon atoms. The presence of such molecular species indicates that asymbiotic gorgonians may use bacterial FAs in biosynthesis of their own wax esters. This observation confirms our hypothesis that bacterial community is important for maintaining the energy balance of azooxanthellate corals.     

Deepwater broadcast spawning by Montastraea cavernosa, Montastraea franksi, and Diploria strigosa at the Flower Garden Banks, Gulf of Mexico

Broadcast spawning by corals is a tightly synchronized process characterized by co-ordinated gamete release within 30–60 min time windows once per year. In shallow water corals, annual water temperature cycles set the month, lunar periodicity the day, and sunset time the hour of spawning. This tight temporal regulation is critical for achieving high fertilization rates in a pelagic environment. Given the differences in light and temperature that occur with depth and the importance of these parameters in regulating spawn timing, it has been unclear whether deeper coral can respond to the same environmental cues that regulate spawning behaviour in shallower coral. In this report, a remotely operated vehicle was used to monitor coral spawning activity at the Flower Garden Banks at depths from 33 to 45 m. Three species Montastraea cavernosa, Montastraea franksi, and Diploria strigosa were documented spawning within this depth range. All recorded spawning events were within the same temporal windows as shallower conspecifics. These data indicate that deep corals at this location either sense the same environmental parameters, despite local attenuation, or communicate with shallower colonies that can sense such spawning cues.     

Carbon translocation from symbiont to host depends on irradiance and food availability in the tropical coral Stylophora pistillata

Reef-building corals live in symbiosis with dinoflagellates that translocate a large proportion of their photosynthetically fixed carbon compounds to their coral host for its own metabolism. The carbon budget and translocation rate, however, vary depending on environmental conditions, coral host species, and symbiont clade. To quantify variability in carbon translocation in response to environmental conditions, this study assessed the effect of two different irradiance levels (120 and 250 μmol photons m^?2 s^?1) and feeding regimes (fed with Artemia salina nauplii and unfed) on the carbon budget of the tropical coral Stylophora pistillata. For this purpose, H¹³CO₃ ^?-enriched seawater was used to trace the conversion of photosynthetic carbon into symbiont and coral biomass and excrete particulate organic carbon. Results showed that carbon translocation (ca. 78 %) and utilization were similar under both irradiance levels for unfed colonies. In contrast, carbon utilization by fed colonies was dependent on the growth irradiance. Under low irradiance, heterotrophy was accompanied by lower carbon translocation (71 %), higher host and symbiont biomass, and higher calcification rates. Under high irradiance, heterotrophy was accompanied by higher rates of photosynthesis, respiration, and carbon translocation (90 %) as well as higher host biomass. Hence, levels of resource sharing within coral–dinoflagellate symbioses depend critically on environmental conditions.     

Changes in morphology and abundance of Stylophora pistillata and Acropora variabilis (anthozoa,hexactiniaria) with respect to depth at Elat,Red Sea

Entire colonies of Stylophora pistillata and Acropora variabilis were collected along a depth transect 100 m south of the Marine Biological Laboratory of Hebrew University Elat, Israel. The branch height of Acropora variabilis decreases significantly with respect to depth, and this species is equitably distributed from 5 to 30 m. Stylophora pistillata demonstrated no decrease in branch height with respect to depth and its distribution is limited to shallow water. The changes in morphology and distribution enhance the credibility of a suggestion made by Roos (1967) that corals common at all depths would show changes in morphology that minimized self-shading. Stylophora pistillata demonstrated an increase in branch density with respect to depth. A hypothesis, based upon the occurrence of Lithophaga cumingiana and water turbulence, is presented to explain the increase in branch density with respect to depth of Stylophora pistillata.     

Incident light and morphology determine coral productivity along a shallow to mesophotic depth gradient

1. While the effects of irradiance on coral productivity are well known, corals along a shallow to mesophotic depth gradient (10–100 m) experience incident irradiances determined by the optical properties of the water column, coral morphology, and reef topography.
2. Modeling of productivity (i.e., carbon fixation) using empirical data shows that hemispherical colonies photosynthetically fix significantly greater amounts of carbon across all depths, and throughout the day, compared with plating and branching morphologies. In addition, topography (i.e., substrate angle) further influences the rate of productivity of corals but does not change the hierarchy of coral morphologies relative to productivity.
3. The differences in primary productivity for different coral morphologies are not, however, entirely consistent with the known ecological distributions of these coral morphotypes in the mesophotic zone as plating corals often become the dominant morphotype with increasing depth.
4. Other colony‐specific features such as skeletal scattering of light, Symbiodiniaceae species, package effect, or tissue thickness contribute to the variability in the ecological distributions of morphotypes over the depth gradient and are captured in the metric known as the minimum quantum requirements.
5. Coral morphology is a strong proximate cause for the observed differences in productivity, with secondary effects of reef topography on incident irradiances, and subsequently the community structure of mesophotic corals.

     

Experimental fragmentation reduces sexual reproductive output by the reef-building coral Pocillopora damicornis

Natural and anthropogenic disturbances may fragment stony reef corals, but few quantitative data exist on the impacts of skeletal fragmentation on sexual reproduction in corals. We experimentally fragmented colonies of the branching coral Pocillopora damicornis and determined the number and size of planula larvae released during one lunar reproductive cycle. Partially fragmented colonies significantly delayed both the onset and peak period of planula release compared with intact control colonies. Most fragments removed from the corals died within 11–18 days, and released few planulae. The total number of planulae released per coral colony varied exponentially with remaining tissue volume, and was significantly lower in damaged versus undamaged colonies. However, the number of planulae produced per unit tissue volume, and planula size, did not vary with damage treatment. We conclude that even partial fragmentation of P. damicornis colonies (<25% of tissue removed) decreases their larval output by reducing reproductive tissue volume. Repeated breakage of corals, such as caused by intensive diving tourism or frequent storms, may lead to substantially reduced sexual reproduction. Therefore, reef management should limit human activities that fracture stony corals and lead to decreases in colony size and reproductive output. Accepted: 2 February 2000     

Age and growth of the cold-water scleractinian Solenosmilia variabilis and its reef on SW Pacific seamounts

Little is known about growth rates of deep-water reef-forming corals or the rates at which these reefs accumulate. Such information is critical for determining the resilience of the reefs to anthropogenic impacts such as trawling and climate change. We radiocarbon date live-caught and sub-fossil samples of the bioherm-forming coral Solenosmilia variabilis collected from precisely known depths and locations by means of a remotely operated vehicle on seamounts south of Tasmania, Australia. The growth rate of colonies live-caught between 958 and 1,454 m, which spans most of the depth range of the species locally, ranged from 0.84 to 1.25 mm linear extension yr^?1 and tended to be higher in the deeper-caught material. Analysis of skeletal microstructure suggests annual deposition of growth increments near the growing tips, but not closer to the base, as the skeleton is extended and thickened. Dating of sub-fossil material indicates S. variabilis has been present on Tasmanian seamounts for at least the last 47,000 yrs and a reef accumulation rate of 0.27 mm yr^?1.     

Effects of intermittent flow and irradiance level on back reef Porites corals at elevated seawater temperatures

Corals inhabiting shallow back reef habitats are often simultaneously exposed to elevated seawater temperatures and high irradiance levels, conditions known to cause coral bleaching. Water flow in many tropical back reef systems is tidally influenced, resulting in semi-diurnal or diurnal flow patterns. Controlled experiments were conducted to test effects of semi-diurnally intermittent water flow on photoinhibition and bleaching of the corals Porites lobata and P. cylindrica kept at elevated seawater temperatures and different irradiance levels. All coral colonies were collected from a shallow back reef pool on Ofu Island, American Samoa. In the high irradiance experiments, photoinhibition and bleaching were less for both species in the intermittent high-low flow treatment than in the constant low flow treatment. In the low irradiance experiments, there were no differences in photoinhibition or bleaching for either species between the flow treatments, despite continuously elevated seawater temperatures. These results suggest that intermittent flow associated with semi-diurnal tides, and low irradiances caused by turbidity or shading, may reduce photoinhibition and bleaching of back reef corals during warming events.     

Symbiodinium genotypic and environmental controls on lipids in reef building corals

Background

Lipids in reef building corals can be divided into two classes; non-polar storage lipids, e.g. wax esters and triglycerides, and polar structural lipids, e.g. phospholipids and cholesterol. Differences among algal endosymbiont types are known to have important influences on processes including growth and the photobiology of scleractinian corals yet very little is known about the role of symbiont types on lipid energy reserves.

Methodology/Principal Findings

The ratio of storage lipid and structural lipid fractions of Scott Reef corals were determined by thin layer chromatography. The lipid fraction ratio varied with depth and depended on symbiont type harboured by two corals (Seriatopora hystrix and Pachyseris speciosa). S. hystrix colonies associated with Symbiodinium C1 or C1/C# at deep depths (>23 m) had lower lipid fraction ratios (i.e. approximately equal parts of storage and structural lipids) than those with Symbiodinium D1 in shallow depths (<23 m), which had higher lipid fraction ratios (i.e. approximately double amounts of storage relative to structural lipid). Further, there was a non-linear relationship between the lipid fraction ratio and depth for S. hystrix with a modal peak at ∼23 m coinciding with the same depth as the shift from clade D to C types. In contrast, the proportional relationship between the lipid fraction ratio and depth for P. speciosa, which exhibited high specificity for Symbiodinium C3 like across the depth gradient, was indicative of greater amounts of storage lipids contained in the deep colonies.

Conclusions/Significance

This study has demonstrated that Symbiodinium exert significant controls over the quality of coral energy reserves over a large-scale depth gradient. We conclude that the competitive advantages and metabolic costs that arise from flexible associations with divergent symbiont types are offset by energetic trade-offs for the coral host.     

Effects of corallivory and coral colony density on coral growth and survival

A suite of processes drive variation in coral populations in space and time, yet our understanding of how variation in coral density affects coral performance is limited. Theory predicts that reductions in density can send coral populations into a predator pit, where concentrated corallivory maintains corals at low densities. In reality, how variation in coral density alters corallivory rates is poorly resolved. Here, we experimentally quantified the effects of corallivory and coral density on growth and survival of small colonies of the staghorn coral Acropora pulchra. Our findings suggest that coral density and corallivory have strong but independent effects on coral performance. In the presence of corallivores, corals suffered high but density-independent mortality. When corallivores were excluded, however, vertical extension rates of colonies increased with increasing densities. While we found no evidence for a predator pit, our results suggest that spatio-temporal variation in corallivore and coral densities can fundamentally alter population dynamics via strong effects on juvenile corals.

     

Ecology of cryptic coral reef communities. III. Abundance and aggregation of encrusting organisms with particular reference to cheilostome bryozoa

The distribution and abundance of encrusting cheilostome bryozoans under foliaceous reef corals was studied at two depths at Rio Bueno, Jamaica. 46 species of cheilostomes were found, but most of these are rare. Only three species occupy, on average, > 5 % of the space available, and each of these is distributed differently. Steginoporella sp. nov. is most abundant at ? 10 m depth where it occasionally overgrows the entire edge zone (marginal 5 cm) of individual corals. Stylopoma spongites (Pallas) is most abundant at ? 21 m where it may also dominate coral edges. Abundance of both these species drops off sharply with distance from coral edges. The third species, Reptadeonella “plagiopora”, is more abundant at ? 10 m than ?21 m depth, but shows no significant variation in cover away from coral edges.Distributions of major groups of encrusting organisms and cheilostome species under corals are predictable enough for definition of spatially distinct assemblages by discriminant function analysis. Particularly distinct is the edge-zone community at ? 10 m where cheilostomes predominate. Nevertheless, variances in organism abundances are extremely high, especially for Steginoporella sp. nov. and Stylopoma spongites the great abundance of which results from relatively few, larger-than-average size colonies, rather than many smaller colonies. Edge-zone communities from replicate m² quadrats at the same depth were significantly different. The same was true within individual quadrats. Transects only 10 to 20 cm apart routinely differed as much as those 50 to 100 m apart. Edge-zone communities also varied significantly with coral size.Increase of Steginoporella sp. nov. and Stylopoma spongites abundance with increasing coral size and age suggests that larval recruitment and juvenile survival by these species is extremely slow and patchy. But once they have reached some critical size, colonies of these species may persist indefinitely by continued clonal growth onto newly grown coral undersurface as it becomes available. Communities under individual corals may develop largely independently, influenced by their unique histories (priority effects) and interactions between their particular inhabitants.     

Tiger cowrie Cypraea tigris feeds on coral-competing sponge Rhabdastrella globostellata in an Acropora dominated reef of Gulf of Mannar,India

Gulf of Mannar (GoM) in the southeast coast of India is known for its coral reefs and reef-associated biodiversity. Corals in GoM were affected to a significant extent by climate change-driven coral bleaching in 2016, and are currently recovering. After the bleaching mortality that corals suffered, the competition for space between corals and sponges is obvious in GoM. Rhabdastrella globostellata is a common marine sponge found overgrowing live coral colonies of the patch reefs in GoM at Pattinamaruthoor in March 2019. Underwater assessment of the reef revealed that 60.06% live coral cover was dominated by Acropora corals (81.91%). Among the acroporans 8.23% of colonies were found overgrown by R. globostellata. During the night dives the tiger cowrie Cypraea tigris was observed to feed on R. globostellata. From this observation the present study infers that C. tigris helps the corals fight these sponges, and concludes that tiger cowries should be protected and promoted to tackle climate change implications.     

The importance of zooplankton to the daily metabolic carbon requirements of healthy and bleached corals at two depths

Bleached and non-bleached fragments of three species of Hawaiian corals were exposed to enhanced and ambient concentrations of zooplankton at 1 and 6 m depth to determine the contribution of zooplankton to the coral's daily carbon budget. The size and taxonomic grouping were recorded for every zooplankton captured and the relative input of zooplankton of different size classes was determined. The contribution of heterotrophy to animal respiration (CHAR) was calculated using an improved method that included the proportionate contribution of zooplankton from all size classes. Results show that the proportionate effects of species, depth and bleaching treatments on coral feeding rates were not significantly different between ambient and enhanced zooplankton concentrations. Corals captured the same size and assemblage of zooplankton under all evaluated conditions, and preferentially captured plankters smaller than 400 µm. Feeding rates of Porites lobata increased with depth regardless of bleaching status. Feeding rates of Porites compressa increased with depth in non-bleached corals, but not in bleached corals. Within depth, feeding rates of bleached Montipora capitata increased, P. compressa decreased and P. lobata remained unchanged relative to non-bleached fragments. Therefore, the feeding response of corals to the same disturbance may vary considerably. Calculated CHAR values show that heterotrophic carbon from zooplankton plays a much larger role in the daily carbon budget of corals than previously estimated, accounting for 46% of some coral species' daily metabolic carbon requirements when healthy and 147% when bleached. Thus, heterotrophically acquired carbon made an important contribution to the daily carbon budget of corals under all experimental conditions. These results suggest that the relative importance of autotrophic and heterotrophic carbon to a coral's energetic needs is mediated by a coral's bleaching status and environment, and should be considered on a continuum, from 100% photoautotrophy to 100% heterotrophy.     

Structure and growth rates of the high-latitude coral: <Emphasis Type="Italic">Plesiastrea versipora</Emphasis>

The high-latitude coral species Plesiastrea versipora was investigated to identify growth rates in colonies over 1 m in diameter. Six colonies from two temperate gulfs (latitudes of 33°–35°S) in Southern Australia were examined using X-ray, luminescence and ²³⁸U/²³⁰Th dating techniques. Annual density bands were present in each coral but varied in width and definition, suggesting different linear extension and calcification rates. Differences in density band width were observed at the local scale (amongst colonies on the same reef) and regional scales (between the two gulfs). Extension rates of the P. versipora colonies examined in this study varied between 1.2 and 7 mm per year, which are amongst the slowest growth rates reported for hermatypic corals. As only one of the six P. versipora colonies had obvious luminescent banding, we conclude that luminescent banding is not an accurate chronological marker in this species of temperate water coral. Coral age estimates derived from counting density bands in X-radiographs ranged from 90 to 320 years for the six colonies studied. U-Th ages from the same colonies determined by high-precision multi-collector inductively coupled plasma mass spectrometer established radiometric ages between 105 and 381 years. The chronological variation in absolute ages between the two techniques varied between 2 and 19% in different colonies, with the lowest growth rates (~1 mm) displaying the greatest variation between density band age and radiometric U-Th age. This result implies that the age of P. versipora and other slow-growing corals cannot be determined accurately from density bands alone. The outcome of this research demonstrates that P. versipora may be useful as a paleoclimate archive, recording several centuries in a single colony in high-latitude environments (corals found in latitudes greater than 30° in either hemisphere), where other well-established coral climate archives, such as Porites, do not occur.     

Chronic low-level nutrient enrichment benefits coral thermal performance in a fore reef habitat

Global- and local-scale anthropogenic stressors have been the main drivers of coral reef decline, causing shifts in coral reef community composition and ecosystem functioning. Excess nutrient enrichment can make corals more vulnerable to ocean warming by suppressing calcification and reducing photosynthetic performance. However, in some environments, corals can exhibit higher growth rates and thermal performance in response to nutrient enrichment. In this study, we measured how chronic nutrient enrichment at low concentrations affected coral physiology, including endosymbiont and coral host response variables, and holobiont metabolic responses of Pocillopora spp. colonies in Mo'orea, French Polynesia. We experimentally enriched corals with dissolved inorganic nitrogen and phosphate for 15 months on an oligotrophic fore reef in Mo'orea. We first characterized symbiont and coral physiological traits due to enrichment and then used thermal performance curves to quantify the relationship between metabolic rates and temperature for experimentally enriched and control coral colonies. We found that endosymbiont densities and total tissue biomass were 54% and 22% higher in nutrient-enriched corals, respectively, relative to controls. Algal endosymbiont nitrogen content cell⁻¹ was 44% lower in enriched corals relative to the control colonies. In addition, thermal performance metrics indicated that the maximal rate of performance for gross photosynthesis was 29% higher and the rate of oxygen evolution at a reference temperature (26.8 °C) for gross photosynthesis was 33% higher in enriched colonies compared to the control colonies. These differences were not attributed to symbiont community composition between corals in different treatments, as C42, a symbiont type in the Cladocopium genus, was the dominant endosymbiont type found in all corals. Together, our results show that in an oligotrophic fore reef environment, nutrient enrichment can cause changes in coral endosymbiont physiology that increase the performance of the coral holobiont.

     

Effects of disturbance on coral communities: bleaching in Moorea,French Polynesia

This study examines patterns of susceptibility and short-term recovery of corals from bleaching. A mass coral bleaching event began in March, 1991 on reefs in Moorea, French Polynesia and affected corals on the shallow barrier reef and to >20 m depth on the outer forereef slope. There were significant differences in the effect of the bleaching among common coral genera, with Acropora, Montastrea, Montipora, and Pocillopora more affected than Porites, Pavona, leptastrea or Millepora. Individual colonies of the common species of Acropora and Pocillopora were marked and their fate assessed on a subsequent survey in August, 1991 to determine rates of recovery and mortality. Ninety-six percent of Acropora spp. showed some degree of bleaching compared to 76% of Pocillopora spp. From March to August mortality of bleached colonies of Pocillopora was 17%, 38% recovered completely, and many suffered some partial mortality of the tissue. In contrast, 63% of the Acropora spp. died, and about 10% recovered completely. Generally, those colonies with less than 50% of the colony area affected by the bleaching recovered at a higher rate than did those with more severe bleaching. Changes in community composition four months after the event began included a significant decrease only in crustose algae and an increase in cover of filamentous algae, much of which occupied plate-like and branching corals that had died in the bleaching event. Total coral cover and cover of susceptible coral genera had declined, but not significantly, after the event.     

Effects of nutrient enrichment on the release of dissolved organic carbon and nitrogen by the scleractinian coral Montipora digitata

The effects of nutrient enrichment on the release of dissolved organic carbon and nitrogen (DOC and DON, respectively) from the coral Montipora digitata were investigated in the laboratory. Nitrate (NO₃ ⁻) and phosphate (PO₄ ³⁻) were supplied to the aquarium to get the final concentrations of 10 and 0.5 μmol l⁻¹, respectively, and the corals were incubated for 8 days. The release rate of DON per unit coral surface area significantly decreased after the nutrient enrichment, while the release rate of DOC was constant. Because the chlorophyll a (chl a) content of zooxanthellae per unit surface area increased, the release rate of DOC significantly decreased when normalized to unit chl a. These results suggested that the incorporation of NO₃ ⁻ and PO₄ ³⁻ stimulated the synthesis of new cellular components in the coral colonies and consequently, reduced extracellular release of DOC and DON. Actually, significant increase in N and P contents relative to C content was observed in the coral’s tissue after the nutrient enrichment. The present study has concluded that inorganic nutrient enrichment not only affects coral-algal metabolism inside the colony but also affects a microbial community around the coral because the organic matter released from corals functions as energy carrier in the coral reef ecosystem.     

Diversity and spatial pattern of coral communities in the Red Sea upper twilight zone

Summary This is the first study based on numerical analysis of the abundance of 11 scleractinian corals of depths at between 100–210 m in the Red Sea twilight zone. Two distinct coral communities were found: a Leptoseris fragilis community at a depth of 100–130 m (zone 1) and a Dendrophillia horsti community below 130 m (zone 2, 3). Population densities and coral coverage are very low; distribution of individuals is highly clumped. Highest observed densities on 100 m² were 2720 individuals for L. fraglis, 2720 for D. horsti and 2260 for Javania insignis. Calculated coverage rates were maximally 3.6% (L. fragilis), 0.08% (D. horsti) and 0.11% (J. insignis). L. fragilis, the only symbiont bearing coral, was very abundant. It has an unusual depth range for a photosynthesising coral. Coral density is only weakly correlated with hard bottom coverage. Species diversity with an average of 8 species is highest at 120–170 m and decreases in shallower and deeper water. The study depth range is a transient zone for coral distribution. It contains the upper distribution limits of a few deep sea corals and the lower ones of several shallower water species. Ahermatypic corals, collected at 160–170 m depth, were transplanted from their original depth to 159, 118, 70 and 40 m; after one year most species survived transplantation far beyond their upper distributional limits. The symbiotic L. fragilis, collected at 120 m, survived transplantation to deep water (159 m) as well as shallow zones (90, 70 and 40 m). The study demonstrates the feasibility of line-transect methods for coral community studies with a submersible.     

Carbonate production by scleractinian corals at Aqaba,Gulf of Aqaba,Red Sea

Summary In a fringing reef at Aqaba at the northern end of the Gulf of Aqaba (29°26′N) growth rates, density, and the calcification rate ofPorites were investigated in order to establish calculations of gross carbonate production for the reefs in this area. Colony accretion ofPorites decreases with depth as a function of decreasing growth rates. The calcification rate ofPorites is highest in shallow water (0–5 m depth) with 0.9 g·cm⁻²·yr⁻¹ and falls down to 0.5 g·cm⁻²·yr⁻¹ below 30 m. Scleractinian coral gross production is calculated from potential productivity and coral coverage. It is mainly dependent on living coral cover and to a lesser extent on potential productivity. Total carbonate production on the reef ranged from 0 to 2.7 kg/m² per year, with a reef-wide average of 1.6 kg/m² perycar. Maximum gross carbonate production by corals at Aqaba occurs at the reef crest and in the middle fore-reef from 10 to 15 m water depth. Production is low in sandy reef parts. Below 30 m depth values still reach ca. 50% of shallow water values. Mean potential production of colonies and gross carbonate production of the whole reef community at Aqaba is lower than in tropical reefs. However, carbonate production is higher than in reef areas at the same latitude in the Pacific, indicating a northward shift of reef production in the Red Sea.