Predicting the Range of Chinese Mitten Crabs in Europe

Abstract:  Ecological niche modeling provides a means for predicting the potential future distribution of a nonindigenous species based on environmental characteristics of the species' native range. We applied this method to the Chinese mitten crab (Eriocheir sinensis) , a catadromous crustacean with a long history of invasion in Europe. We used genetic algorithm for rule-set prediction to predict the potential European distribution of mitten crab based on its distribution in 42 locations in its native Asia. The climatic variables, air temperature, number of days, amount of precipitation, and wetness index, contributed significantly to predictions of native distribution limits. Although the genetic algorithm for rule-set prediction model was developed for the native range, the species' extensive distribution in Europe ( n = 434) allowed independent validation of the predictions. Application of the model to Europe was successful, with 84% of occurrences in regions predicted to be suitable by >80% of the models and <4% of occurrences in areas predicted suitable by <50% of the models (mainly along the northern range). At the watershed scale, areas with established mitten crab populations had significantly higher habitat matching than sites that were not invaded. The independent validation of the Asian-based model by the European distribution revealed that predictions were highly accurate. The model also identified large areas of Europe, particularly along the Mediterranean coast, as vulnerable to future invasion. These predictions can be used to develop strategies to control the spread of mitten crab by preventing introductions into vulnerable areas.     

Genetic diversity and population structure of the Chinese mitten crab <Emphasis Type="Italic">Eriocheir sinensis</Emphasis> in its native range

The Chinese mitten crab Eriocheir sinensis is an indigenous and economically important species in China, but can also be found as invasive species in Europe and America. Mitten crabs have been exploited extensively as a food resource since the 1990s. Despite its ecological and economic importance, the genetic structure of native mitten crab populations is not well understood. In this paper, we investigated the genetic structure of mitten crab populations in China by screening samples from ten locations covering six river systems at six microsatellite loci. Our results provide further evidence that mitten crabs from the River Nanliujiang in Southern China are a genetically differentiated population within the native range of Eriocheir, and should be recognized as a separate taxonomic unit. In contrast, extremely low levels of genetic differentiation and no significant geographic population structure were found among the samples located north of the River Nanliujiang. Based on the reproductive biology of mitten crabs and the geography of their habitat we argue that both natural and human-mediated gene flow are unlikely to fully account for the similar allele frequency distributions at microsatellite loci. Large population sizes of mitten crabs suggest instead that a virtual absence of genetic drift and significant homoplasy of microsatellite alleles have contributed to the observed pattern. Furthermore, a coalescent-based maximum likelihood method indicated a more than two-fold lower effective population size of the Southern population compared to the Northern Group and low but significant levels of gene flow between both areas.     

Bridging information domains to improve ecological understanding of biological invasions in a marine ecosystem

We devised a practical method for integrating information on 2 marine invasive species using 3 different approaches: standardized ecological monitoring, online-reporting databases, and surveys of anglers and crabbers. Focusing on 2 recently introduced species with different characteristics, the Asian shore crab (Hemigrapsus sanguineus) and Chinese mitten crab (Eriocheir sinensis), in the Hudson-Raritan watershed of New York and New Jersey, we used sensitivity analyses to explore the relative contribution of each information source to knowledge of species abundance and distribution. All 3 information sources contributed something unique to understanding abundance and distribution of the introduced crabs. Online and survey data on Asian shore crabs significantly affected predictions of abundance, whereas monitoring data did not. When survey data were omitted, abundance estimates were unchanged over time, but when they were included, the model predicted an increased abundance in 2012. All 3 data sets for the Asian shore crab significantly affected estimates of species coverage; surveys had the biggest influence, increasing range size by 4097.25 km². For the catadromous Chinese mitten crab, ecological monitoring data collected in freshwater shortly after the original sighting significantly shaped model estimates for abundance and documented the establishment phase of the mitten crab in an area outside the spatial scope of the surveyed resource users. However, the survey data significantly enlarged mitten crab range-size estimates by 6498.01 km². By demonstrating that data integration produced an image of the invasion process that would not have emerged had we used any 1 method individually, model results provide evidence for the advantages of an interdisciplinary approach.     

Introduction,dispersal and potential impacts of the green crab Carcinus maenas in San Francisco Bay,California

The North Atlantic portunid crab Carcinus maenas (Linnaeus, 1758) has invaded the North Pacific Ocean following more than two centuries of global dispersal due to human activities. C. maenas was first collected in San Francisco Bay, California, in 1989–1990, where its distribution and prey selectivity were investigated in 1992–1994. It has become abundant in shallow, warm lagoons (which as favorable and retentive microhabitats may have served as invasion incubators) and spread throughout the north, central and south bays. It may have arrived in ballast water, on fouled ships, amongst algae with imported live bait or lobsters, or by intentional release; genetic comparisons of the Bay population with possible source populations may aid in defining the transport mechanism. C. maenas' eurytopic nature, its high breeding potential, and its diet and feeding behavior suggest the potential for extensive ecosystem alterations through predator-prey interactions, competition, disturbance, and indirect effects. Although both negative economic impacts through reduction or disruption of fisheries and positive impacts of providing bait and human-food fisheries have been documented in a few regions, the potential economic impacts in San Francisco Bay remain largely unknown.     

Conservation implications of density-dependent predation by ghost crabs on hatchling sea turtles running the gauntlet to the sea

Protecting eggs from predators is common practice in sea turtle conservation, but routine protection of hatchlings is not. Of 42 loggerhead hatchlings observed emerging from 10 nests on undeveloped Onslow Beach, North Carolina, 24 % were preyed on by ghost crabs. In experimental trials, ghost crabs similarly threatened and captured neonate freshwater sliders, supporting their substitution as proxy for threatened and endangered sea turtle hatchlings in field experiments testing density dependence. Exploiting natural long-shore variation in ghost crab density, we show that a 2.6-fold higher ghost crab density resulted in 5 times more nocturnal threat encounters with sliders and 3.4 times more slider captures. Sliders released in simulated group emergences experienced lower per capita capture risk by ghost crabs than solitary sliders, implying predator dilution. Non-independence of egg and hatchling depredation motivates consideration of merging sea turtle egg and hatchling stages when modeling and managing food web interactions.     

Fish and ships: relating dispersal frequency to success in biological invasions

 Most studies characterizing successful biological invaders emphasize those traits that help a species establish a new population. Invasions are, however, multi-phase processes with at least two phases, dispersal and introduction, that occur before establishment. Characteristics that enhance survival at any of these three phases will contribute to invasion success. Here, we synthesize information on the dispersal, introduction, and establishment of fishes mediated by ship ballast-water transport. We synthesize 54 reports of at least 31 fish species collected from ballast tanks (Phase 1), including 28 new reports from our recent studies (1986 to 1996). Our literature survey revealed 40 reports of 32 fish species whose introductions have been attributed to ballast transport (Phase 2), of which at least 24 survived to establish persistent populations (Phase 3). We detected little overlap at the species level between these two data sets (Phase 1 vs Phases 2 and 3), but patterns emerged at the family level. The Gobiidae (6 species), Clupeidae (4 species), and Gasterosteidae (1 species) were the most commonly found fish families in ballast tanks (Phase 1). The Gobiidae (13 species), Blenniidae (6 species) and Pleuronectidae (2 species) dominated the list of ballast-mediated introductions (Phase 2); gobies and blennies were the families most frequently established (Phase 3). The invasive success of gobies and blennies may be explained in part by their crevicolous nature: both groups seek refuge and lay eggs in small holes, and may take advantage of the ballast-intake holes on ship hulls. This behavior, not typically associated with invasive ability, may contribute to successful introduction and establishment by facilitating the dispersal phase of invasion. The failure of the pleuronectids to invade may reflect poor salinity match between donor and recipient regions. To develop a predictive framework of invasion success, organisms must be sampled at all three phases of the invasion process. Our comparison of two ballast sampling methods suggests that fishes have been undersampled in ballast-water studies, including our own, and that the role of ballast transport in promoting fish invasions has been underestimated. Received: 13 January 1999 / Accepted: 24 February 2000     

Fecundity as a Basis for Risk Assessment of Nonindigenous Freshwater Molluscs

Abstract:  The most efficient way to reduce future damages from nonindigenous species is to prevent the introduction of harmful species. Although ecologists have long sought to predict the identity of such species, recent methodological advances promise success where previous attempts failed. We applied recently developed risk assessment approaches to nonindigenous freshwater molluscs at two geographic scales: the Laurentian Great Lakes basin and the 48 contiguous states of the United States. We used data on natural history and biogeography to discriminate between established freshwater molluscs that are benign and those that constitute nuisances (i.e., cause environmental and/or economic damage). Two statistical techniques, logistic regression and categorical tree analysis, showed that nuisance status was positively associated with fecundity. Other aspects of natural history and biogeography did not significantly affect likelihood of becoming a nuisance. We then used the derived statistical models to predict the chance that 15 mollusc species not yet in natural ecosystems would cause damage if they become established. We also tested whether time since establishment is related to the likelihood that nonindigenous mollusc species in the Great Lakes and United States would cause negative impacts. No significant relationship was evident at the U.S. scale, but recently established molluscs within the Great Lakes were more likely to cause negative impacts. This may reflect changing environmental conditions, changing patterns of trade, or may be an indication of "invasional meltdown." Our quantitative analyses could be extended to other taxa and ecosystems and offer a number of improvements over the qualitative risk assessments currently used by U.S. (and other) government agencies.     

Aktive Stoffe als Handlungsoption gegen die Verschleppung von Organismen durch Seeschiffe?

Background and Scope

The marine shipping lanes have become the most important path for the invasion of foreign aquatic organisms. The increasing global trade results in an increase in the number of marine vessels. Without any protective measures, with every ship carrying foreign organisms the risk of biological invasions will rise.

Results

The International Martime Organization (IMO) of the United Nations developed a convention to reduce the transfer of organisms with ballast water. After the entry-into-force of this convention the fleet of the world has to be equipped with effective ballast water management technology before 2016. This article discusses potential options for action. To comply with the limit values of the ballast water convention, the current state of the art demands the use of active substances. Any decision on the approval of active substances used for ballast water treatment and the environmental impact assessment lies with the IMO. Proposed to day are UV-radiation, active chlorine, active oxygen, the creation of biocides through electrolysis and a change in gases contained in the ballast water. The technologies and the potential risks are presented.

Conclusions

Despite the introduction of an approval procedure by IMO any reliable assessment of the real risks involved in the use of biocides is impossible, as the risk assessment approaches have still to be developed. On the regulatory level, the main focus in data requirements for the risk assessment is on a comprehensive testing of the toxic potential of the biocides proposed. Strategies for the identification and evaluation of the chemical resulting from the treatment of limnic, marine and brackish water are not fully developed. An integrating assessment of risks involved in the introduction of foreign organisms versus the toxic effects of the substances used or created during treatment is still missing.     

Cryptic invasion of northeast Pacific estuaries by the Asian polychaete, Hediste diadroma (Nereididae)

The estuarine polychaete, Hediste diadroma (Nereididae), of eastern Asia was found at all six sites sampled in October 2009 in northeast Pacific estuaries, including the Columbia River, along a 300-km stretch of coastline in Washington and Oregon, USA. The immature worms were returned to the laboratory in Japan where 13 of them were cultured to sexual maturity after 5–7 months. These worms had the diagnostic, epitoke-specific spinigers that distinguish H. diadroma from all other Hediste species. Their egg sizes (140–170 μm), paragnath numbers on the proboscis, and chromosome number (2n = 28) were also consistent with those previously reported for the Japanese population of H. diadroma. The widespread distribution of H. diadroma indicates that it is a successful invader of the North American Pacific and it may have long escaped detection because of its morphological similarity to the native species, H. limnicola. The long pelagic life of H. diadroma larvae could have facilitated its successful trans-ocean dispersal with ballast water of ships.     

Partitioning vegetation response to anthropogenic stress to develop multi-taxa wetland indicators.

Emergent plants can be suitable indicators of anthropogenic stress in coastal wetlands if their responses to natural environmental variation can be parsed from their responses to human activities in and around wetlands. We used hierarchical partitioning to evaluate the independent influence of geomorphology, geography, and anthropogenic stress on common wetland plants of the U.S. Great Lakes coast and developed multi-taxa models indicating wetland condition. A seven-taxon model predicted condition relative to watershed-derived anthropogenic stress, and a four-taxon model predicted condition relative to within-wetland anthropogenic stressors that modified hydrology. The Great Lake on which the wetlands occurred explained an average of about half the variation in species cover, and subdividing the data by lake allowed us to remove that source of variation. We developed lake-specific multi-taxa models for all of the Great Lakes except Lake Ontario, which had no plant species with significant independent effects of anthropogenic stress. Plant responses were both positive (increasing cover with stress) and negative (decreasing cover with stress), and plant taxa incorporated into the lake-specific models differed by Great Lake. The resulting models require information on only a few taxa, rather than all plant species within a wetland, making them easier to implement than existing indicators.     

Predicting regional spread of non-native species using oceanographic models: validation and identification of gaps

Predicting spread is a central goal of invasion ecology. Within marine systems, researchers have increasingly made use of oceanographic circulation models to estimate currents and track species dispersal. However, the accuracy of these models for predicting biological patterns, particularly for non-native species, has generally not been validated. Particularly, we wished to examine the ability of models to predict physical and biological processes, which jointly determine the spread of marine larval organisms. We conducted two empirical studies—a recruitment study and a drift card study—along the coast of New England, USA, focusing on two invaders of concern—the European green crab (Carcinus maenas) and the Asian shore crab (Hemigrapsus sanguineus), to explicitly evaluate the ability of oceanographic models to predict patterns of spread. We used data from the large-scale drift card study to validate our ability to capture dispersal patterns driven purely by physical processes. Next, we conducted a recruitment study to evaluate our ability to reproduce patterns of biological dispersal. We were generally capable of reproducing drift cards patterns—suggesting that the physical mechanics in the model were predictive. However, predicted biological patterns were inconsistent—we were able to predict dispersal patterns for H. sanguineus but not for C. maenas. Our results highlight the importance of validating models and suggest that more work is necessary before we can reliably use oceanographic models to predict biological spread of intertidal organisms.     

Resource degradation: a subtle effect of bottom fishing

Populations of hermit crabs are critically limited by the availability of suitable gastropod shells that they utilise to reduce their risk of predation and environmental stress. Common whelks are the main source of shells for large hermit crabs in the northern Atlantic but are vulnerable to direct and indirect effects of fishing activity. This study examined the potential consequences of degrading shell resources for common hermit crabs. Laboratory trials demonstrated that hermit crabs avoid low-quality damaged shells throughout their life history. This laboratory preference was corroborated by direct field observations of shells preferentially occupied by hermit crabs, compared with shells available for occupation. In the field, 8 times as many empty shells had holes compared to shells occupied by hermit crabs. In the North Sea, the abundance and biomass of live whelks and hermit crabs collected at sites where they co-occurred were significantly related. However, whelks occurred at far fewer sites overall and were more patchily distributed at high abundance than hermit crabs, which were more widespread. At a subset of sites, whelks of the same body-mass range occurred in the Irish and North Sea. However, at these sites, hermit crabs sampled from the North Sea had a significantly lower biomass. This suggests that the shells available for occupation at the North Sea sites would not support crabs of a body mass comparable to that found in the Irish Sea. Using published data, we calculated that in some of the intensively fished areas of the North Sea, 24% of the available shell resource will be damaged each year. The reduction in shell quality in the North Sea may impose a physical constraint on the upper size limit currently attainable by hermit crabs and hence may have implications for population viability.Communicated by J. P. Thorpe, Port Erin     

Biotic resistance to green crab, <Emphasis Type="Italic">Carcinus maenas</Emphasis>, in California bays

The distribution of the introduced European green crab, Carcinus maenas, was investigated in the central California embayments of Bodega Bay Harbor (BBH), Tomales Bay, and Bolinas Lagoon using baited traps and snorkel surveys. Adult green crabs were very spatially limited in all three embayments and occurred primarily in warm, shallow areas that lacked large native Cancer spp. crabs. The green crabs that were found in closest proximity to populations of Cancer spp. exhibited high levels of limb damage and loss; damage was strongly correlated with low ratios of intertidal area: edge, indicative of narrow areas of intertidal that are more easily accessed by large Cancer spp. moving up to forage during periods of tidal inundation. Up to 70% of the green crabs tethered in areas of BBH that are utilized by Cancer spp. experienced limb loss, while those tethered in the marsh, where there are no Cancer spp., were undamaged. The results suggest that the potential distribution of green crabs in the northeastern Pacific will be far less than has been predicted, and that their impacts may be largely attenuated through predation by and competition with native crab species.     

Reproductive cycles in two populations of the Pacific sand dollar Dendraster excentricus

East Pacific swimming crabs are poorly known ecologically. We trawled samples along 4 estuarine-continental shelf transects to provide information on the population structure and species composition of portunids along environmental stress gradients. Portunids comprised almost 99% of the benthic crabs. Callinectes toxotes dominates the freshest estuarine areas, but is replaced by C. arcuatus in estuary mouth and shallow-shelf samples. Deeper shelf samples are dominated by Euphylax robustus and Portunus sper. Great numbers of E. dovii, a pelagic species, were taken on the shelf; their size distribution implies intraspecific competition. Evidence is given that differences in salinity tolerance account for differential landward limits, but competition, predation by fishes, increased fouling and decreased available food exclude eurytopic species from physicochemically equable shelf waters.     

Shell-size selection by intertidal sympatric hermit crabs

This study evaluated selection for shell size by three species of tropical intertidal hermit crabs, Clibanarius antillensis, C. sclopetarius, and C. vittatus, from species of shells which are frequently used in nature. Crab size and weight were strongly and significantly related to all measured parameters of the selected shells. The strength of these relationships (r² values) depended neither on the crab nor on the shell variables taken into account. The relationships between crab size and the dimensions of the selected shells showed higher r² values than the corresponding relationships with the shells that the crabs had occupied when they were collected (0.482–0.903 in comparison to 0.091–0.652, respectively), indicating that the crabs were occupying sub-optimal shells in nature. Negative allometry was frequently found in the relationships between crab and shell variables, indicating that large crabs select and use proportionally lighter shells than do small crabs. This negative allometry was stronger for the shells used in nature (except for C. antillensis), i.e. larger crabs tended to select heavier shells in the laboratory than in nature. Different allometric relationships were also recorded among the dimensions of shells used in nature and those selected by the hermit crabs in free-access experiments: as shell length increased, the selected shells were heavier and had larger apertures than the shells used in nature. The relationships between crab size and the length and weight of the selected shells did not depend on the species of crab or species of shell, but only on crab size. Therefore, analyses using these variables can be performed without taking the species of crab or shell into account, i.e. data from different crab or shell species can be pooled. The influence of crab and/or shell species was recorded only in the models fitted for aperture length and width, variables which were more related to shell architecture than did shell length or weight. In contrast, if crab weight is used as an independent variable, different crab or shell species can be analyzed together independently of the particular shell parameter. This indicates that crab weight may be less susceptible than crab shield length to shell morphological constraints. Finally, the results indicate that the preferred size of a given shell type chosen by a given hermit crab will depend more on crab size or weight, than on the crab or shell species under consideration, i.e. crab shell-size relationships are not species specific.Communicated by P.W. Sammarco, Chauvin     

Trait‐based prediction of extinction risk of small‐bodied freshwater fishes

Small body size is generally correlated with r‐selected life‐history traits, including early maturation, short‐generation times, and rapid growth rates, that result in high population turnover and a reduced risk of extinction. Unlike other classes of vertebrates, however, small freshwater fishes appear to have an equal or greater risk of extinction than large fishes. We explored whether particular traits explain the International Union for Conservation of Nature (IUCN) Red List conservation status of small‐bodied freshwater fishes from 4 temperate river basins: Murray‐Darling, Australia; Danube, Europe; Mississippi‐Missouri, North America; and the Rio Grande, North America. Twenty‐three ecological and life‐history traits were collated for all 171 freshwater fishes of ≤120 mm total length. We used generalized linear mixed‐effects models to assess which combination of the 23 traits best explained whether a species was threatened or not threatened. We used the best models to predict the probability of 29 unclassified species being listed as threatened. With and without controlling for phylogeny at the family level, small body size—among small‐bodied species—was the most influential trait correlated with threatened species listings. The k‐folds cross‐validation demonstrated that body size and a random effect structure that included family predicted the threat status with an accuracy of 78% (SE 0.5). We identified 10 species likely to be threatened that are not listed as such on the IUCN Red List. Small body size is not a trait that provides universal resistance to extinction, particularly for vertebrates inhabiting environments affected by extreme habitat loss and fragmentation. We hypothesize that this is because small‐bodied species have smaller home ranges, lower dispersal capabilities, and heightened ecological specialization relative to larger vertebrates. Trait data and further model development are needed to predict the IUCN conservation status of the over 11,000 unclassified freshwater fishes, especially those under threat from proposed dam construction in the world's most biodiverse river basins.     

Description and comparison of major foregut ossicles in hydrothermal vent crabs

Major foregut (gastric mill) ossicles, including the dorsal median tooth, lateral teeth, accessory lateral teeth, and cardiopyloric valve, of hydrothermal vent crabs were dissected and examined during the summer of 1996 from specimens housed at the Natural History Museum of Los Angeles County. Ossicles are described for two species of hydrothermal vent crabs (family Bythograeidae Williams, 1980). The western Pacific Austinograea williamsi Hessler and Martin has an unusual dorsal median tooth. The surrounding cuticular flange is scalloped and bears spinulose setae at the tip of each of the protruding edges, a condition perhaps unique in the Brachyura. The lateral teeth are mostly unremarkable, bearing the typical large anterior denticles and deep serrations seen in other crab families, but with a higher number of serrations than is known for any species previously described. The accessory lateral teeth bear flattened, plate-like spines that are widest basally and that taper to a cylindrical tip. The basic armature of the foregut of Bythograea thermydron Williams, known only from vents in the eastern Pacific, is very similar. Scalloping of the median tooth borders is less pronounced, however, and the shape of the tooth itself and of the plate from which it arises is slightly different. The lateral teeth bear fewer and more widely spaced grooves, and the cardiopyloric valve entrance appears less setose at its extremity. Comparison with foregut ossicles in other crab families based on earlier studies, most of which have not employed SEM, reveals some similarities between bythograeids and some xanthids, but does not clarify the phylogenetic position of the bythograeids. Because of the paucity of other SEM studies of the brachyuran foregut, it is difficult to ascertain whether some of the many spine and setal types in the bythograeid foregut are unique or even unusual compared to those of other crab families. Nothing about the foregut of the vent crabs is indicative of their unusual habitat. Anecdotal observations of feeding in vent crabs indicate that they are opportunistic scavengers and omnivores, which is in keeping with the non-specialized nature of the foregut. The debate between adaptation vs phylogeny as determinants of the form of the gastric mill components is briefly discussed. Received: 10 December 1996 / Accepted: 13 November 1997     

Einige neue Aspekte bei der Osmoregulation von Carcinus maenas

Employing specimens of the decapod crab Carcinus maenas L. from Kiel Bay in the Baltic Sea (～13 to 18‰S) and from Helgoland in the North Sea (～32‰S), comparative studies were made on the osmotic and sodium regulations. After a 3-day exposure to low salinities of 5 or 10‰, the internal medium of the crabs from the Baltic Sea has a greater freezing point depression and a higher sodium content, than that of the North Sea crabs. In normal sea water, the blood of North Sea specimens is slightly hyperosmotic; the blood of specimens from the Baltic Sea is nearly isosmotic to the surrounding medium. Individual cross adaptations to the different habitat salinities (2 to 3 weeks) result in a reduction of the osmotic differences in specimens of the two populations; the differences do not, however, completely disappear.     

Multivariate analysis of the mud crab Scylla serrata (Brachyura: Portunidae) from four locations in Southeast Asia

The large, edible mud crab Scylla serrata (Forskål) exhibits different phenotypes which are recognised by the fishermen of Southeast Asia and are given local names such as “white”, “green”, “red” or “black” crabs on the basis of their colouration. A preliminary study using multivariate techniques was undertaken in order to examine the degree of dissimilarity between the different morphs of commercially fished stocks of S. serrata. Twenty-two morphometric and 20 meristic characters were measured on male crabs from four locations in three countries in Southeast Asia, including Surat Thani in Thailand where “black” and “white” morphs exist sympatrically. Canonical variate analysis (CVA) on the morphometric data discriminated three groups with no chain-linking. Surat Thani “white” crabs and those from south Vietnam were phenotypically similar and formed one distinct group; a second group contained crabs from Ranong (Thailand) and Sarawak which were phenotypically “black”; the third group contained “black” crabs from Surat Thani. CVA on meristic data confirmed two groupings, but implied that the Surat Thani “black” morph may only be a variant of the Surat Thani/Vietnam “white” form, rather than a third species. These findings are discussed in relation to the ecology, fishery management and aquaculture potenial of S. serrata.