Ingenious floral structure drives explosive pollination in Hydrilla verticillata (Hydrocharitaceae).

In explosive pollination, many structures and mechanisms have evolved to achieve high-speed stamen movement. The male flower of the submerged plant Hydrilla verticillata is reported to be able to release pollen explosively some time after leaving the mother plant time, but the mechanism of stamen movement and the related functional structure in this species are unclear. In this study, we observed the male flower structure and pollen dispersal process of H. verticillata. We analysed the stamen movements during the pollen dispersal process and conducted several controlled experiments to study the process of storage and release of elastic potential energy in explosive pollination. When the male flower of H. verticillata is bound to the united bracts, the sepals accumulate elastic potential energy through the expansion of basal extensor cells. After the male flower is liberated from the mother plant, the stamens unfold rapidly with the sepals under adhesion and transfer the elastic potential energy to the filament in seconds. Once stamens unfold to a critical angle, at which the elasticity of the filament just exceeds the adhesion between sepals and anthers, the stamens automatically rebound and release pollen in milliseconds. These results reveal that Catapult-like stamens, spoon-shaped sepals and enclosed united bracts in the spathe together constitute the functional structure in rapid stamen movement of H. verticillata. They ensure that the pollen can be released on the water surface, and thus adapt successfully to the pollen-epihydrophilous pollination.

Biomechanical response of a submerged, rosette-forming macrophyte to wave action in a eutrophic lake on the Yungui Plateau, China.

Few studies have focused on the biomechanical responses of submerged, rosette-forming macrophytes to wave action, water depth, or their co-occurrence in naturally eutrophic systems. The plant architecture, root anchorage strength-related traits, leaf morphology, and biomechanics of Vallisneria natans inhabiting a range of water depths were examined along three transects (T1, T2, and T3) in a eutrophic lake, Lake Erhai, in Yunnan Province, China. These transects were exposed to weak wave action and hyper-eutrophication (T1), moderate wave action and eutrophication (T2), or strong wave action and eutrophication (T3). The results showed that V. natans was mainly distributed at intermediate depths, with the widest colonization depth in T1. The values of plant architecture, root anchorage strength-related traits, leaf morphology, and biomechanics were generally highest in T3 and smallest in T2. Along the depth gradient, these values were generally highest at 3.5, 2.5, and 2.5 m for the plants growing in T1, T2, and T3, respectively. These findings suggest that V. natans adopts a "tolerance" strategy to cope with the effects of strong wave action in eutrophic habitats and an "avoidance" strategy when exposed to moderate wave action in eutrophic areas. Since the absence of an avoidance strategy increases the resistance to low-light stress at the expense of increased drag forces, there is a limit to the wave action that V. natans can withstand. This study indicates that biomechanics could be important when determining the distribution pattern of V. natans in Lake Erhai.

Characterization of phosphorus availability in response to radial oxygen losses in the rhizosphere of Vallisneria spiralis.

The viewpoint that radial oxygen loss (ROL) of submerged macrophytes induces changes in redox conditions and the associated phosphorus (P) availability has been indirectly confirmed at larger spatial scales using conventional, destructive techniques. However, critical information about microniches has largely been overlooked due to the lack of satisfactory in situ mapping technologies. In this study, we deployed a recently developed hybrid sensor in the rhizosphere of Vallisneria spiralis (V. spiralis) during two vegetation periods to provide 2-D imaging of the spatiotemporal co-distribution of oxygen (O2) and P from a fixed observation point. Overall, the images of O2 and P showed a high degree of spatiotemporal heterogeneity throughout the rhizosphere at the sub-mm scale. A clear decrease in the P mobilization corresponded well to the steep O2 enhancement within a 2-mm-thick zone around younger V. spiralis root, indicating a significant coupling relationship between ROL and P availability. Surprisingly, despite significant diurnal shifts in ROL along the older V. spiralis roots, P availability did not fluctuate in a substantial part of the rhizosphere throughout the day; however, ROL increased the P immobilization significantly by changing the redox gradients at the outer rhizosphere. This study clearly demonstrates how continuous ROL of V. spiralis can play a major role in regulating P availability within the rhizosphere. The premise behind this statement is the discovery of how this continuous ROL can lead to the formation of three distinctive redox landscapes in the rooting sediment (oxic, suboxic, or anaerobic layers).

A functional-structural model of ephemeral seagrass growth influenced by environment.

Background and Aims: Ephemeral seagrasses that respond rapidly to environmental changes are important marine habitats. However, they are under threat due to human activity and are logistically difficult and expensive to study. This study aimed to develop a new functional-structural environmentally dependent model of ephemeral seagrass, able to integrate our understanding of ephemeral seagrass growth dynamics and assess options for potential management interventions, such as seagrass transplantation. Methods: A functional-structural plant model was developed in which growth and senescence rates are mechanistically linked to environmental variables. The model was parameterized and validated for a population of Halophila stipulacea in the Persian Gulf. Key Results: There was a good match between empirical and simulated results for the number of apices, net rhizome length or net number of internodes using a 330 d simulation. Simulated data were more variable than empirical data. Simulated structural patterns of seagrass rhizome growth qualitatively matched empirical observations. Conclusions: This new model successfully simulates the environmentally dependent growth and senescence rates of our case-study ephemeral seagrass species. It produces numerical and visual outputs that help synthesize our understanding of how the influence of environmental variables on plant functional processes affects overall growth patterns. The model can also be used to assess the potential outcomes of management interventions like seagrass transplantation, thus providing a useful management tool. It is freely available and easily adapted for new species and locations, although validation with more species and environments is required.

Permanently open stomata of aquatic angiosperms display modified cellulose crystallinity patterns.

Most floating aquatic plants have stomata on their upper leaf surfaces, and usually their stomata are permanently open. We previously identified 3 distinct crystallinity patterns in stomatal cell walls, with angiosperm kidney-shaped stomata having the highest crystallinity in the polar end walls as well as the adjacent polar regions of the guard cells. A numerical bio-mechanical model suggested that the high crystallinity areas are localized to regions where the highest stress is imposed. Here, stomatal cell wall crystallinity was examined in 4 floating plants from 2 different taxa: basal angiosperms from the ANITA grade and monocots. It appears that the non-functional stomata of floating plants display reduced crystallinity in the polar regions as compared with high crystallinity of the ventral (inner) walls. Thus their guard cells are both less flexible and less stress resistant. Our findings suggest that the pattern of cellulose crystallinity in stomata of floating plants from different families was altered as a consequence of similar evolutionary pressures.

Response of Thalassia Testudinum Morphometry and Distribution to Environmental Drivers in a Pristine Tropical Lagoon.

This study was undertaken to determine the relationships between the biomass, morphometry, and density of short shoots (SS) of the tropical seagrass Thalassia testudinum and the physical-environmental forcing in the region. Seasonal sampling surveys were undertaken four times in Bahia de la Ascension, a shallow estuary in the western Mexican Caribbean, to measure plant morphology and environmental variables. The estuary has a fresh water-influenced inner bay, a large central basin and a marine zone featuring a barrier reef at the seaward margin. Leaf size was positively correlated with increasing salinity, but total biomass was not, being similar across most of the sites. Aboveground biomass exhibited seasonal differences in dry and rainy seasons along the bay, most markedly in the brackish inner bay where an abrupt decline in biomass coincided with the rainy season. The relationship between nutrients and biomass indicates that the aboveground/belowground biomass ratio increases as nutrient availability increases. Areal cover was inversely correlated with SS density during both dry and rainy seasons. Maximum SS recruitment coincided with the rainy season. Peaks in SS density were recorded in the freshwater-influenced inner bay during an ENSO cold phase in 2007 ("La Niña") which is associated with a wetter dry season and following a strong storm (Hurricane Dean). The onset of the rainy season influences both shoot density and T. testudinum biomass by controlling the freshwater input to the bay and thus, the system's salinity gradient and external nutrients supply from the coastal wetland.

Effect of nutrient enrichment on the source and composition of sediment organic carbon in tropical seagrass beds in the South China Sea.

To assess the effect of nutrient enrichment on the source and composition of sediment organic carbon (SOC) beneath Thalassia hemprichii and Enhalus acoroides in tropical seagrass beds, Xincun Bay, South China Sea, intertidal sediment, primary producers, and seawater samples were collected. No significant differences on sediment δ(13)C, SOC, and microbial biomass carbon (MBC) were observed between T. hemprichii and E. acoroides. SOC was mainly of autochthonous origin, while the contribution of seagrass to SOC was less than that of suspended particulate organic matter, macroalgae and epiphytes. High nutrient concentrations contributed substantially to SOC of seagrass, macroalgae, and epiphytes. The SOC, MBC, and MBC/SOC ratio in the nearest transect to fish farming were the highest. This suggested a more labile composition of SOC and shorter turnover times in higher nutrient regions. Therefore, the research indicates that nutrient enrichment could enhance plant-derived contributions to SOC and microbial use efficiency.

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa.

BACKGROUND AND AIMS: Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. METHODS: Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. KEY RESULTS: Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. CONCLUSIONS: The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells.

Developing a methodology of bioindication of human-induced effects using seagrass morphological variation in Spermonde Archipelago, South Sulawesi, Indonesia.

Seagrass is particularly susceptible to environmental degradation. The objective of the study is to develop an effective bioindicator to assess human-induced effects using morphological variation and fluctuating asymmetry (FA) of seagrass. Samples were collected from eight islands situated at different distance from mainland with different human population density and therefore expected to experience different level of anthropogenic pressure. Cd, Pb, Cu, Zn, nitrate, and phosphate were measured. Metals were also measured in tissues of seagrass. Metal concentrations in sediment, water, and seagrass did not exceed the quality standards required for marine life. Heterogeneity of FA was found among sites suggesting that there are some factors changing developmental instability of seagrass which is not associated to particular toxicants. This baseline study indicates that the water condition is still natural and shows no signs of metal contamination, therefore it does not cause a detectable stress on morphological variation and FA of seagrass.

Genetic species identification and population structure of Halophila (Hydrocharitaceae) from the Western Pacific to the Eastern Indian Ocean.

BACKGROUND: The Indo-Pacific region has the largest number of seagrass species worldwide and this region is considered as the origin of the Hydrocharitaceae. Halophila ovalis and its closely-related species belonging to the Hydrocharitaceae are well-known as a complex taxonomic challenge mainly due to their high morphological plasticity. The relationship of genetic differentiation and geographic barriers of H. ovalis radiation was not much studied in this region. Are there misidentifications between H. ovalis and its closely related species? Does any taxonomic uncertainty among different populations of H. ovalis persist? Is there any genetic differentiation among populations in the Western Pacific and the Eastern Indian Ocean, which are separated by the Thai-Malay peninsula? Genetic markers can be used to characterize and identify individuals or species and will be used to answer these questions. RESULTS: Phylogenetic analyses of the nuclear ribosomal internal transcribed spacer region based on materials collected from 17 populations in the Western Pacific and the Eastern Indian Ocean showed that some specimens identified as H. ovalis belonged to the H. major clade, also supported by morphological data. Evolutionary divergence between the two clades is between 0.033 and 0.038, much higher than the evolutionary divergence among H. ovalis populations. Eight haplotypes were found; none of the haplotypes from the Western Pacific is found in India and vice versa. Analysis of genetic diversity based on microsatellite analysis revealed that the genetic diversity in the Western Pacific is higher than in the Eastern Indian Ocean. The unrooted neighbor-joining tree among 14 populations from the Western Pacific and the Eastern Indian Ocean showed six groups. The Mantel test results revealed a significant correlation between genetic and geographic distances among populations. Results from band-based and allele frequency-based approaches from Amplified Fragment Length Polymorphism showed that all samples collected from both sides of the Thai-Malay peninsula were clustered into two clades: Gulf of Thailand and Andaman Sea. CONCLUSIONS: Our study documented the new records of H. major for Malaysia and Myanmar. The study also revealed that the Thai-Malay peninsula is a geographic barrier between H. ovalis populations in the Western Pacific and the Eastern Indian Ocean.

Phytogeography of Najas gracillima (Hydrocharitaceae) in North America and its cryptic introduction to California.

PREMISE OF THE STUDY: The discontinuous North American distribution of Najas gracillima has not been explained satisfactorily. Influences of extirpation, nonindigenous introduction, and postglacial migration on its distribution were evaluated using field, fossil, morphological, and molecular data. Najas is a major waterfowl food, and appropriate conservation measures rely on accurate characterization of populations as indigenous or imperiled. • METHODS: Seed lengths of N. gracillima from native Korean populations, a nonindigenous Italian population, and North American populations were compared using digital image analysis. DNA sequence analyses from these regions provided nine nrITS genotypes and eight cpDNA haplotypes. • KEY RESULTS: Najas gracillima seeds from Eurasia and California are shorter than those from eastern North America. Nuclear and chloroplast DNA sequences of N. gracillima from Korea and Italy were identical to California material but differed from native eastern North American plants. Eastern North American specimens of N. gracillima at localities above the last glacial maximum boundary were identical or similar genetically to material from the northeastern United States and Atlantic Coastal Plain and Piedmont but divergent from plants of the Interior Highlands-Mississippi Embayment region. • CONCLUSIONS: In California, N. gracillima is nonindigenous and introduced from Asia. In eastern North America, populations that colonized deglaciated areas were derived primarily from refugia in the Atlantic Coastal Plain and Piedmont. Genetic data indicate initial postglacial migration to northeastern North America, with subsequent westward dispersal into the Upper Great Lakes. These results differentiate potentially invasive California populations from seriously imperiled indigenous eastern North American populations.

Cadmium-induced biochemical responses of Vallisneria spiralis.

The following study was carried out to investigate the cadmium (Cd) accumulating potential of Vallisneria. After subjecting plants to different concentrations of Cd, it was observed that plants are able to accumulate ample amount of metal in their roots (5,542 microg g(-1) dw) and leaves (4,368 microg g(-1) dw) in a concentration- and duration-dependent manner. Thus, it is evident that the accumulation in roots was 1.3 times higher than the shoots. It was also noted that with increasing Cd accumulation, roots of the plant appeared darker in color and harder in texture. In response to metal exposure, amount of low molecular weight antioxidants such as cysteine and nonprotein thiols (NP-SH) and activity of enzymes such as APX and GPX were significantly enhanced at lower concentrations of Cd, followed by decline at higher doses. It was also observed that in exposed plants, activity of APX enzyme was higher in roots (ca. 3 times) as compared to leaves. However, chlorophyll and protein content was found to decline significantly in a dose-dependent manner. Results suggested that due to its high accumulation potential, Vallisneria may be effectively grown in water bodies moderately contaminated with Cd.

Laboratory investigation of the acoustic response of seagrass tissue in the frequency band 0.5-2.5 kHz.

Previous in situ investigations of seagrass have revealed acoustic phenomena that depend on plant density, tissue gas content, and free bubbles produced by photosynthetic activity, but corresponding predictive models that could be used to optimize acoustic remote sensing, shallow water sonar, and mine hunting applications have not appeared. To begin to address this deficiency, low frequency (0.5-2.5 kHz) acoustic laboratory experiments were conducted on three freshly collected Texas Gulf Coast seagrass species. A one-dimensional acoustic resonator technique was used to assess the biomass and effective acoustic properties of the leaves and rhizomes of Thalassia testudinum (turtle grass), Syringodium filiforme (manatee grass), and Halodule wrightii (shoal grass). Independent biomass and gas content estimates were obtained via microscopic cross-section imagery. The acoustic results were compared to model predictions based on Wood's equation for a two-phase medium. The effective sound speed in the plant-filled resonator was strongly dependent on plant biomass, but the Wood's equation model (based on tissue gas content alone) could not predict the effective sound speed for the low irradiance conditions of the experiment, in which no free bubbles were generated by photosynthesis. The results corroborate previously published results obtained in situ for another seagrass species, Posidonia oceanica.

The interaction of CO2 concentration and spatial location on O2 flux and mass transport in the freshwater macrophytes Vallisneria spiralis and V. americana.

The biology of aquatic organisms determines the maximum rates of physiological processes, but the mass transport of nutrients determines the nominal rates at which these processes occur. Maximum O(2) flux (P(max)) at 17.1 mmol m(-3) CO(2) was higher for the leaves of the freshwater macrophyte Vallisneria spiralis [P(max)=0.013+/-0.001 mmol m(-2) s(-1) (g(chla+b) m(-2))(-1) (mean +/- s.e.m.)] than for the closely related species, Vallisneria americana [P(max)=0.008+/-0.001 mmol m(-2) s(-1) (g(chla+b) m(-2))(-1)]. The O(2) flux saturated at freestream velocities >4.5+/-1.2 cm s(-1) and was spatially invariant for both species. However, a tenfold decrease in CO concentration to 1.71 mmol m(-3) changed the nature of the relationship between O(2) flux and spatial location along the leaf surface, and reduced the O(2) flux of V. spiralis to values similar to V. americana. The O(2) flux [P(max)=0.007+/-0.001 mmol m(-2) s(-1) (g(chla+b) m(-2))(-1)] saturated at the upstream location (i.e. 1 cm from the leading edge of the leaf) but was found to increase linearly with freestream velocity [slope=0.057+/-0.011 mmol m(-2) s(-1) (g(chla+b) m(-2))(-1) (m s(-1))(-1)] at the downstream location (i.e. 7 cm from the leading edge) at freestream velocities >1.8+/-0.9 cm s(-1). Conversely, mass transfer rates did not vary with CO(2) concentration, and were characteristic of a laminar concentration boundary layer at the upstream location and a turbulent concentration boundary layer at the downstream location. Rates of mass transfer measured directly from O(2) profiles were not predicted by theoretical values based on hydrodynamic measurements. Moreover, the concentration boundary layer thickness (delta(CBL)) values measured directly from O(2) profiles were 48+/-2% and 21+/-1% of the predicted theoretical delta(CBL) values at the upstream and downstream locations, respectively. It is evident that physiological processes involving mass transport are coupled and vary in space. Mass transport investigations of biological systems based solely on hydrodynamic measurements need to be interpreted with caution.

Mapping invasive aquatic vegetation in the Sacramento-San Joaquin Delta using hyperspectral imagery.

The ecological and economic impacts associated with invasive species are of critical concern to land managers. The ability to map the extent and severity of invasions would be a valuable contribution to management decisions relating to control and monitoring efforts. We investigated the use of hyperspectral imagery for mapping invasive aquatic plant species in the Sacramento-San Joaquin Delta in the Central Valley of California, at two spatial scales. Sixty-four flightlines of HyMap hyperspectral imagery were acquired over the study region covering an area of 2,139 km(2) and field work was conducted to acquire GPS locations of target invasive species. We used spectral mixture analysis to classify two target invasive species; Brazilian waterweed (Egeria densa), a submerged invasive, and water hyacinth (Eichhornia crassipes), a floating emergent invasive. At the relatively fine spatial scale for five sites within the Delta (average size 51 ha) average classification accuracies were 93% for Brazilian waterweed and 73% for water hyacinth. However, at the coarser, Delta-wide scale (177,000 ha) these accuracy results were 29% for Brazilian waterweed and 65% for water hyacinth. The difference in accuracy is likely accounted for by the broad range in water turbidity and tide heights encountered across the Delta. These findings illustrate that hyperspectral imagery is a promising tool for discriminating target invasive species within the Sacramento-San Joaquin Delta waterways although more work is needed to develop classification tools that function under changing environmental conditions.

Form-function analysis of the effect of canopy morphology on leaf self-shading in the seagrass Thalassia testudinum.

The variation in seagrass morphology and the magnitude of leaf self-shading within the canopy of Thalassia testudinum, were compared among nine sites in a fringing reef lagoon. We found a significant variation in the growth-form of T. testudinum reflected in a 5.4-fold variation in the attenuation coefficient (K (d)) within the canopy. The largest morphological variation was observed in shoot density. Leaf biomass, leaf area index (LAI), and shoot density were positively associated with canopy-K (d) and with the percentage of surface irradiance received by the top of the seagrass canopy (% Es). These results provide an explanation for the consistent pattern of depth reduction in seagrass leaf biomass and shoot density reported in the literature. Shoot density and shoot size are two descriptors of the growth-form of T. testudinum related to its clonal life-form. Shoot size was not significantly correlated with canopy-K (d), nevertheless, it showed a significant effect on the slope of the relationship between shoot density and canopy-K (d). According to this model, shoot size also contributes to light attenuation within the seagrass canopy by increasing the effect of shoot density. This form-function analysis suggests that light may have a relevant role in the regulation of the optimal plant balance between horizontal (variation in shoot density) and vertical (variation in shoot size) growth of seagrasses. Other environmental factors and interactions also need to be examined to fully understand the mechanistic bases of the morphological responses of seagrasses to the environment.

The structure and productivity of the Thalassia testudinum community in Bon Accord Lagoon, Tobago

The Thalassia testudinum dominated seagrass community in the Buccoo Reef/ Bon Accord Lagoon Marine Park, measures 0.5 km(2) and is part of a contiguous coral reef, seagrass bed and mangrove swamp system in southwest Tobago. 7 testudinum coverage, productivity and percent turnover rates were measured from February 1998 to February 1999 at four sample locations, while total T. testudinum biomass was measured at two locations in the lagoon from 1992-2002. Productivity and turnover rates varied spatially and seasonally. They were higher in the back-reef area than in the mangrove-fringed lagoon, and were lowest at locations near to a sewage outfall. 7 testudinum coverage ranged from 6.6% in the lagoon to 68.5% in the back-reef area while productivity ranged from 3.9 to 4.9 g dry wt m(-2) d(-1) . Productivity and percentage turnover rates were higher in the dry season (January-June) than in the wet season (July-December). Productivity ranged from 3.0 in the wet season to 5.0 g dry wt m2 d-' in the dry season while percentage turnover rates ranged from 4.2% to 5.6%. Total Thalassia biomass and productivity in Bon Accord Lagoon were compared to six similar sites in the Caribbean that also participate in the Caribbean Coastal Marine Productivity Program (CARICOMP). This seagrass community is being negatively impacted by nutrient-enriched conditions

The structure and productivity of the Thalassia testudinum community in Bon Accord Lagoon, Tobago.

The Thalassia testudinum dominated seagrass community in the Buccoo Reef/ Bon Accord Lagoon Marine Park, measures 0.5 km(2) and is part of a contiguous coral reef, seagrass bed and mangrove swamp system in southwest Tobago. 7 testudinum coverage, productivity and percent turnover rates were measured from February 1998 to February 1999 at four sample locations, while total T. testudinum biomass was measured at two locations in the lagoon from 1992-2002. Productivity and turnover rates varied spatially and seasonally. They were higher in the back-reef area than in the mangrove-fringed lagoon, and were lowest at locations near to a sewage outfall. 7 testudinum coverage ranged from 6.6% in the lagoon to 68.5% in the back-reef area while productivity ranged from 3.9 to 4.9 g dry wt m(-2) d(-1) . Productivity and percentage turnover rates were higher in the dry season (January-June) than in the wet season (July-December). Productivity ranged from 3.0 in the wet season to 5.0 g dry wt m2 d-' in the dry season while percentage turnover rates ranged from 4.2% to 5.6%. Total Thalassia biomass and productivity in Bon Accord Lagoon were compared to six similar sites in the Caribbean that also participate in the Caribbean Coastal Marine Productivity Program (CARICOMP). This seagrass community is being negatively impacted by nutrient-enriched conditions.