Using otolith microchemical analysis to investigate the importance of brackish bays for pike (Esox lucius Linnaeus, 1758) reproduction in the southern Baltic Sea

The brackish Baltic Sea is inhabited by several primary freshwater fish species. The pike (Esox lucius) is the largest of these species and acts as the top predator in the marine food web of the Baltic Sea. Prior studies have shown the importance of brackish bays as spawning grounds for the Baltic Sea pike. To identify the spawning habitat of the pike population around the island of Rügen (southern Baltic Sea)—either freshwater streams flowing into the Baltic or brackish lagoons of the Baltic—we used the ICPMS method to quantify the strontium to calcium (Sr:Ca) ratios in sagittal otoliths of 79 adult pike. In total, only six per cent (n = 5) of the analysed Baltic Sea individuals were classified as being of freshwater origin. Ninety‐four per cent (n = 74) of analysed pike were classified as having hatched in brackish water. Compared to previous studies on Baltic Sea pike, our sample shows a high proportion of pike that is of brackish origin: this highlights the adaptive capacity of this species with regard to its ability to reproduce in different salinities. Our data highlight the importance of brackish bays for pike reproduction in the southern Baltic Sea, even if pike live on the upper limit of their salinity tolerance in this area. This is an important finding considering decreases in commercial catches in the last 30 years indicating a population decline supposedly related to recruitment failures.     

Effects of salinity on growth and mortality of migratory and resident forms of Eurasian perch in the Baltic Sea

Abstract – In the coastal areas of the Baltic Sea, there are two forms of perch (Perca fluviatilis). One of the forms is migratory and spawns in streams entering the Baltic Sea. The other form is resident and spawns in brackish water. Both forms utilise the coastal habitat for foraging. We examined the spawning success of the two forms in fresh and brackish water (7‰, equal to salinity in the south Baltic Sea). The experiments showed that hatching success was equally high in freshwater and in brackish water despite female origin. The survival of yellow‐sac and free swimming fry was significantly reduced in brackish water, which was independent if the fish was of migratory or brackish resident origin. Further, growth rate of perch fry was severely reduced in brackish water. The results indicate that perch has not developed any tolerance to brackish water in the young life stages. The migratory life strategy of perch can thus be explained by higher survival of fry in freshwater.     

Genetic population structure of pike (Esox lucius Linnaeus, 1758) in the brackish lagoons of the southern Baltic Sea

The Baltic Sea is a unique ecosystem and the largest body of brackish water in the world. It is inhabited by several originally freshwater fish species, one of them being pike (Esox lucius), which is adapted to the mesohaline conditions of the southern Baltic Sea and colonises its south‐western bays and lagoons. Using twelve microsatellite markers, we analysed the genetic structure of 407 Baltic Sea pike collected from ten locations in the coastal zone of the southern Baltic region and adjacent rivers and nearby lakes. We used traditional F‐statistics and a Bayesian approach implemented in STRUCTURE to investigate spatial structuring. Our results show an overall isolation by distance pattern with two distinct coastal clusters. The border area between the two clusters is characterised by a sharp salinity leap, indicating that the dispersal barrier is not physical, but physiological in nature. A third cluster was formed by three lake populations, presumably as a result of past stocking measures. Additional otolith microchemical studies demonstrate the brackish origin and residency of the majority of the pike from the coastal lagoons, supporting previous studies which suggest that the anadromous pike “ecotype” is scarce in this area of the Baltic Sea. Our results contribute to current knowledge on the population genetics of Baltic Sea pike in a previously unsampled area.     

Elemental analysis of otoliths, fin rays and scales: a comparison of bony structures to provide population and life-history information for the Arctic grayling (Thymallus arcticus)

Abstract –  We used laser ablation inductively coupled plasma mass spectrometry line scans to determine the elemental composition of otoliths, pectoral fin rays and scales of Arctic grayling. Elemental signatures of otoliths and pectoral fin rays effectively provide life-history information on individual fish, important for management of grayling, and potentially all freshwater teleosts. Bulk elemental signatures measured in the otoliths and fin rays were highly correlated to the stream chemistries where the fish were captured. A surprising result of this study was that fin rays showed the strongest relationship with water chemistry for strontium. Scale strontium concentrations were not correlated to water chemistries suggesting that other physiological mechanisms, or remobilisation, may be influencing the deposition of trace elements within scales. Linear discriminant function analyses for otolith and fin ray elemental signatures (and intriguingly also for scales) separated fish from different rivers for all three structures; thus, this technique can be used effectively as a means to discriminate origin of capture.     

Do Eurasian minnows (Phoxinus phoxinus L.) inhabiting brackish water enter fresh water to reproduce: Evidence from a study on otolith microchemistry

Dynamics of coastal fish assemblages in the Baltic Sea are still rather poorly understood. In particular, little information is available on migrations and movements of the small‐bodied littoral fish species like Eurasian minnow (Phoxinus phoxinus L.). Minnow is considered typically as a river and lake species, but it also inhabits brackish coastal waters of the Baltic Sea. In this study, we investigated movement patterns of brackish water inhabiting minnows using otolith microchemistry. Fish were collected from four different sites (from two bays and two stream mouths) around Saaremaa Island. The results indicated that at least three distinct migration patterns exist: (i) seawater residency, (ii) fast springtime migrations to fresh water or to bays with lower salinities (areas near stream mouths) and (iii) prolonged migrations to fresh water (some cases overwintering in streams). Migration patterns listed above were not evenly distributed among sites, and some individuals did not migrate to fresh water in every year. None of the analysed fish were freshwater residents. Additionally, potentially ontogenetic effects on Mn and Sr concentrations were observed in the otolith core regions, which may have important implications for the interpretation of otolith chemistry data. The overall findings of this study demonstrate that much more complex migration patterns exists in brackish water inhabiting minnow populations than previously thought and at least some individuals do enter freshwater to reproduce.     

Getting cosy in freshwater: Assumed to be brackish pike are not so brackish after all

Pike (Esox lucius) occupy coastal streams and rivers of the Baltic Sea, where they attain large sizes (>5 kg). These large sizes are perhaps due to the fact that they can tolerate relatively high salinities and can thus forage in the nearby more productive brackish environments. In an attempt to quantify the extent to which pike utilise brackish environments, and to provide some insight into the underlying causes for brackish water migrations, we tagged 30 pike from a western Baltic river with acoustic transmitters and were able to track 21 individuals for 1 year. Based on experienced from local anglers, this population was assumed to be brackish in nature, where individuals underwent freshwater migrations to spawn. Our findings however suggest that the smallest and most active individuals make short exits into brackish waters and do so on rare occasions. Our results further indicate that neither sex nor size is related to activity level. We suggest that these patterns reflect two distinct behaviours—active and passive—and that large pike can be supported by the food availability in the river, without the need to venture into coastal zones, thus defying the conventional view that Baltic pike are all brackish in nature.     

Annual and seasonal dynamics of fish in the brackish-water Matsalu Bay, Estonia

Abstract –  Matsalu Bay in the Väinameri (West-Estonian Archipelago Sea) is a relatively shallow but large bay. It is the only real delta estuary in the northern Baltic Sea. The fish fauna is strongly dominated by freshwater species such as perch Perca fluviatilis , roach Rutilus rutilus , white bream Blicca bjoerkna and rudd Scardinius erythrophthalmus . The study presents CPUE data describing annual and seasonal variations in the species composition, abundance and spatial distribution, based on 1 year of monthly samplings from August 2001 to August 2002 and routine coastal monitoring (in late July–early August) between 1994 and 2003. The bay can be divided into three rather distinct parts. The fish fauna of the inner part (salinity 0–2 p.p.t.) is different from two open parts and resembles that of freshwater lakes. In the openmost part (salinity usually 4–6 p.p.t.) some marine species, such as herring Clupea harengus membras , flounder Platichthys flesus and eelpout Zoarces viviparus may seasonally be abundant. Species composition and abundance of most species varies seasonally and interannually. Only the abundance of few species such as pike Esox lucius and pikeperch Stizostedion lucioperca was not impacted by seasonality. The most dynamic period in the bay is spring, when several species (some of them very rare in summer, such as herring and smelt Osmerus eperlanus ) enter the bay for spawning.     

Otolith elemental characteristics of whitefish (Coregonus lavaretus) from brackish waters of the Gulf of Bothnia,Baltic Sea

To investigate whether European whitefish (Coregonus lavaretus) groups in the brackish (≤7%) Gulf of Bothnia (Baltic Sea) express specific otolith elemental characteristics, concentrations of elements (Ca, Ba, Sr, Zn, Mn, Fe) in whole otoliths were studied by inductively coupled plasma‐optical emission spectrometry. Whitefish (N = 67) were sampled from six different latitudinal sites; four in the sea and two in rivers along the west coast of Finland, and from a fresh water lake for comparison. The concentration of several elements showed significant differences among sampling sites. Otolith Ba concentrations were higher at the northern sampling sites compared to the southern ones, thereby showing negative association to capture‐habitat salinity. In contrast, otolith Sr and Zn concentrations were lower at the northern sampling sites, thereby associating positively to capture‐habitat salinity. Otolith Mn concentrations did not associate with latitude or salinity but markedly varied between sampling sites. Elevated Fe concentrations occurred in fish otoliths from the two northernmost sites. Ca concentrations were at the same levels in otoliths from all whitefish samples. Otoliths of whitefish from the lake had very high Ba and low Sr concentrations compared to concentrations in otoliths from whitefish in the sea and rivers. Our results show that otolith elemental characteristics of whitefish vary considerably at various geographic scales showing high potential for population identification using elemental fingerprints. Analysis of otolith elemental characteristics may therefore provide an efficient tool for the identification of main areas of residence, spawning locations, and migration routes, thus contributing towards the sustainable management of whitefish stocks.     

High salinity tolerance in eggs and fry of a brackish Esox lucius population

Abstract Knowledge on the biology and physiology of pike, Esox lucius L., populations inhabiting saline environments is scarce. An experimental setup was used to examine egg development and fry behaviour and growth under varying salinity levels in a brackish‐water pike population from the western Baltic Sea. Eggs and fry developed at 8.5 psu, which is higher than hitherto reported for other populations. Fry exhibited stress behaviour and reduced growth when subjected to salinities above 13 psu. This indicates that early life stages of E. lucius tolerate ambient salinity conditions equivalent to the natural environment in its nursery area in the Stege Nor lagoon. The population should therefore not be dependent on access to freshwater environments during spawning. However, the results also suggest that the population’s natural environment is on the margin of its physiological capacity. The results have significance for management of brackish‐water E. lucius populations, e.g. in relation to stocking strategies.     

Vertical movements of coastal pike (Esox lucius)—On the role of sun basking

Knowledge of patterns and drivers of the spatiotemporal distribution of top predatory fish is key to understand ecological dynamics and to successful management. Here, we integrated field and laboratory approaches to study vertical movements of pike (Esox lucius) in relation to season, light regimes and body temperature. We tagged pike from the Baltic Sea with data storage tags during spawning migration and retrieved them during migration the following years to obtain high-resolution data from full year of movements. The results showed seasonal and diel patterns of activity and body temperature that conformed to distinct patterns of crepuscular activity and diel vertical migrations. The latter manifested as two different patterns, either a stationary phase in the surface water during day followed by night-time in deeper water or vice versa. The occurrence of these two behaviours varied among individuals and within individuals among seasons. Diel vertical migration has previously not been described for this shallow-dwelling species, but was a common and consistent behaviour among individuals in this study. We suggest that the function of the daytime surface behaviour in pike is to increase body temperature through sun basking. This thermoregulatory role of surfacing was supported by the laboratory study where individuals sought the surface layer, exposed themselves to infrared light and thereby attained body temperatures in excess of ambient water. These results support sun basking as a mechanism for heat gain and further suggest that access to sunlight in the surface layer could be an important driver of vertical migrations.     

Elemental analysis of otoliths of Japanese anchovy: trial to discriminate between Seto Inland Sea and Pacific stock

The ability to discriminate local stocks of Japanese anchovy Engraulis japonicus was assessed based on data from four elements (K, Na, P, and Sr) using an electron probe micro analyzer (EPMA) and data from three elements (Ba, Mn, and Sr) using inductively coupled plasma mass spectrometry (ICP-MS) from the otoliths of 40 anchovy (23.6–47.0 mm body length). Anchovy were caught at three sites (Aki-nada, Hiuchi-nada, and Osaka Bay) in the Seto Inland Sea, and one site (Kuroshio extension) in the Pacific Ocean in 2002. In order to discriminate different spawning grounds, EPMA data from the core portion (from core to 30 μm in the core-posterior axis) were used. Results showed that it was difficult to discriminate between the Seto Inland Sea and the Pacific anchovy by EPMA data. Conversely, it was possible to discriminate between the Seto Inland Sea and the Pacific anchovy by ICP-MS data from bulk otoliths. Our results showed that Mn contents of otoliths using ICP-MS discriminate between spawning grounds most, and Ba and Sr discriminate less. The difference in elemental compositions in anchovy otoliths between the Seto Inland Sea and the Pacific Ocean might be reflected by cumulative experienced elemental composition of ambient sea water during life history between the Seto Inland Sea and the Pacific anchovy.     

The effect of lakes on the stream fish fauna

The effect of natural lakes on the fish species distribution in surrounding streams was studied using electrofishing survey data from 3281 localities in streams throughout Sweden. The occurrence of lake fish (northern pike, European perch, roach and burbot) was significantly higher close to lakes, both up- and downstream, as compared to localities further up- or downstream of lakes. Correspondingly, the occurrences of stream fish (brown trout, grayling, European minnow and bullheads) were lower close to lakes. It is suggested that the lower occurrences of the latter close to lakes be due to biotic interaction with lake fish, especially predation from pike and burbot. The results strongly indicate that the effect of lakes on water temperature, drift of plankton and invertebrates or the moderating effect on water fluctuations and quality did not influence the stream fish fauna to the same extent as the presence of lake fish in a zone up- as downstream of the lakes. Lakes function as a reservoir of strong predators and competitors.     

Identification of hatchery-reared and naturally produced Atlantic salmon, Salmo salar L., juveniles based on examination of otoliths

. Atlantic salmon, Salmo salar L., parr and smolts from three Norwegian rivers were examined with respect to whether or not they had been released from a hatchery. The hatchery background was known for some released fish and could be ascertained for others from their aberrant body morphology and eroded fins. Fish released after one winter in a hatchery had opaque otoliths like the fish examined from two hatcheries. Naturally produced fish showed a distinct, seasonal growth pattern in the otoliths, alternating between opaque summer zones and hyaline winter zones. Fish released as fry showed an otolith pattern similar to that of naturally produced fish. A test revealed little discrepancy between two independent otolith readings even though the test reading was conducted without any information about the fish accompanying the otoliths. The results suggest that examination of otoliths may help distinguish between juveniles that are hatchery-reared and juveniles that are naturally produced in the river, provided that the hatchery-reared fish have a 1-year history in the hatchery prior to release.     

Effect of salinity on strontium:calcium ratios in the otoliths of Sakhalin taimen, <Emphasis Type="Italic">Hucho perryi</Emphasis>

In order to determine the salinity effect on otolith strontium (Sr):calcium (Ca) ratios, the Sr and Ca contents of otoliths of Sakhalin taimen, Hucho perryi, reared for 60 days in four salinities (freshwater, one-third seawater, two-thirds seawater, and full seawater) were examined. The Ca and Sr contents and the resultant Sr:Ca ratios in the rearing water increased linearly with salinity. Otolith Sr:Ca ratios were also found to be positively correlated with salinity. However, the Sr:Ca did not correspond to temperature fluctuations during the experiment. These results indicate that Sr:Ca ratios in otoliths can be used to reconstruct the migratory history of the fish by differentiating when the fish migrated between freshwater and seawater.     

Whole otolith elemental analysis reveals feeding migration patterns causing growth rate differences in anadromous whitefish from the Baltic Sea

Impact of feeding migration pattern on growth rate of whitefish (Coregonus lavaretus L.) from River Kemijoki was investigated. River Kemijoki is located in the northernmost Gulf of Bothnia (Baltic Sea). Whitefish ascending the river to spawn show major growth rate variations. To study whether these variations are related to different feeding areas (migratory connectivity), whole otoliths from fast‐growing (FAST^G) whitefish (n = 9, age 4 years, average weight 790 g) and slowly growing (SLOWLY^G) whitefish (n = 9, age 8 years, average weight 600 g) were analysed for elemental (Ba, Sr, Mg, Zn, Mn, Ca) concentrations by inductively coupled plasma‐optical emission spectrometry. Results showed that the FAST^G whitefish had much lower otolith Ba concentration (~45%, p ≤ .01) than SLOWLY^G whitefish, revealing that FAST^G whitefish have spent more time in water of higher salinity/lower latitude, that is at feeding grounds in the southern Gulf of Bothnia. Otoliths of the FAST^G whitefish had also higher Mg (~25%, p ≤ .001) and Zn (~20%, p ≤ .05) concentrations, but lower Ca (~5%, p ≤ .01) concentrations, than the otoliths from SLOWLY^G whitefish. These results further reveal that FAST^G and SLOWLY^G whitefish have been spatially separated. In conclusion, bulk otolith elemental analysis revealed spatial separation and migration differences in river‐spawning whitefish subpopulations in the Gulf of Bothnia, which apparently lead to differences in fish growth rate. Our results increase the understanding of river‐spawning whitefish biology and its population structure and dynamics and may contribute to a sustainable management of river‐spawning whitefish stocks.     

A comparative study of juvenile salmon density in 20 streams throughout a very large river system in northern Norway

Abstract – The objective was to compare juvenile salmon density in 20 streams throughout the very large River Tana, northern Norway, and to relate variation in density to a suite of environmental factors. Four sampling sites were electrofished in each stream (one at the mouth of the stream and three within the stream) in August and October 2000, 2001, 2002. 0+ salmon parr were absent from seven streams, present at the mouth of 11 streams, and present within only two streams, both of which were probably spawning streams. Older parr migrated upstream into most streams and their highest densities were usually found in streams flowing directly into the spawning habitat in the three largest tributaries of the Tana or the river itself. Juvenile salmon were sparse or absent in streams flowing into smaller tributaries. Most streams with high parr densities were those of dense riparian vegetation that provided terrestrial invertebrates as drift food for the salmon parr, cover for fish, cooler stream temperatures in summer, and food for benthic stream invertebrates that were also a source of food for the parr.     

Behaviour,escapement and mortality of female European silver eels within a regulated lowland river draining into the Baltic Sea

Migration patterns of 146 female silver eels on their way through fresh and brackish water stretches of the Warnow River into the Baltic Sea were studied using acoustic telemetry. The majority of eels (77%) regained their expected downstream migration behaviour after a median rehabilitation time of 2 days. Positive rheotactic behaviour was observed for 23% of tagged eels after release, probably influenced by experimental procedures. The observed silver eel escapement rate was 63%. Two migratory groups were classified by significantly different migratory speed, time of residence in brackish waters and biometric silvering characteristics. Although the classification of silvering stages is based on these measures, the proportions of these stages did not reflect the observed differences in migratory behaviour. In the context of a permanent silver eel descent in the freshwater part of the Warnow River, a permanent escapement was likewise occurring into the Baltic Sea.     

Atlantic salmon Salmo salar L., brown trout Salmo trutta L. and Arctic charr Salvelinus alpinus (L.): a review of aspects of their life histories

Abstract – Among the species in the family Salmonidae, those represented by the genera Salmo, Salvelinus, and Oncorhynchus (subfamily Salmoninae) are the most studied. Here, various aspects of phenotypic and life‐history variation of Atlantic salmon Salmo salar L., brown trout Salmo trutta L., and Arctic charr Salvelinus alpinus (L.) are reviewed. While many strategies and tactics are commonly used by these species, there are also differences in their ecology and population dynamics that result in a variety of interesting and diverse topics that are challenging for future research. Atlantic salmon display considerable phenotypic plasticity and variability in life‐history characters ranging from fully freshwater resident forms, where females can mature at approximately 10 cm in length, to anadromous populations characterised by 3–5 sea‐winter (5SW) salmon. Even within simple 1SW populations, 20 or more spawning life‐history types can be identified. Juveniles in freshwater can use both fluvial and lacustrine habitats for rearing, and while most smolts migrate to sea during the spring, fall migrations occur in some populations. At sea, some salmon undertake extensive oceanic migrations while other populations stay within the geographical confines of areas such as the Baltic Sea. At the other extreme are those that reside in estuaries and return to freshwater to spawn after spending only a few months at sea. The review of information on the diversity of life‐history forms is related to conservation aspects associated with Atlantic salmon populations and current trends in abundance and survival. Brown trout is indigenous to Europe, North Africa and western Asia, but was introduced into at least 24 countries outside Europe and now has a world‐wide distribution. It exploits both fresh and salt waters for feeding and spawning (brackish), and populations are often partially migratory. One part of the population leaves and feeds elsewhere, while another part stays as residents. In large, complex systems, the species is polymorphic with different size morphs in the various parts of the habitat. Brown trout feed close to the surface and near shore, but large individuals may move far offshore. The species exhibits ontogenetic niche shifts partly related to size and partly to developmental rate. They switch when the amount of surplus energy available for growth becomes small with fast growers being younger and smaller fish than slow growers. Brown trout is an opportunistic carnivore, but individuals specialise at least temporarily on particular food items; insect larvae are important for the young in streams, while littoral epibenthos in lakes and fish are most important for large trout. The sexes differ in resource use and size. Females are more inclined than males to become migratory and feed in pelagic waters. Males exploit running water, near‐shore and surface waters more than females. Therefore, females feed more on zooplankton and exhibit a more uniform phenotype than males. The Arctic charr is the northernmost freshwater fish on earth, with a circumpolar distribution in the Holarctic that matches the last glaciation. Recent mtDNA studies indicate that there are five phylogeographic lineages (Atlantic, Arctic, Bering, Siberian and Acadian) that may be of Pleistocene origin. Phenotypic expression and ecology are more variable in charr than in most fish. Weights at maturation range from 3 g to 12 kg. Population differences in morphology and coloration are large and can have some genetic basis. Charr live in streams, at sea and in all habitats of oligotrophic lakes, including very deep areas. Ontogenetic habitat shifts between lacustrine habitats are common. The charr feed on all major prey types of streams, lakes and near‐shore marine habitats, but has high niche flexibility in competition. Cannibalism is expressed in several cases, and can be important for developing and maintaining bimodal size distributions. Anadromy is found in the northern part of its range and involves about 40, but sometimes more days in the sea. All charr overwinter in freshwater. Partial migration is common, but the degree of anadromy varies greatly among populations. The food at sea includes zooplankton and pelagic fish, but also epibenthos. Polymorphism and sympatric morphs are much studied. As a prominent fish of glaciated lakes, charr is an important species for studying ecological speciation by the combination of field studies and experiments, particularly in the fields of morphometric heterochrony and comparative behaviour.     

Spatial integration of freshwater fish markets in the Northern Baltic Sea area

Freshwater fish species and Baltic salmon (Salmo salar) are important to small-scale fisheries in Finland and Sweden. The formerly local markets for these species have expanded as trade has been opened up to international competition. In this study we use cointegration analysis to test the spatial integration of freshwater fish markets in Finland and between Finland and Sweden. The analysed fish species are salmon, perch (Perca fluviatilis), pikeperch (Sander lucioperca), European whitefish (Goregonus lavaretus) and pike (Exos lucius), and the data covers ex-vessel prices from 1993 or 1995 to 2004. We found that the regional prices in Finland were cointegrated. This indicates that the prices are determined on a single market in Finland. Moreover, the study suggests that Finnish and Swedish markets in Baltic salmon, whitefish, pikeperch and perch were partially integrated, while integration of pike markets could not be found. The political implication is that an essential part of the local small-scale fisheries’ operational environment is determined outside the national borders.     

Landscape features determine brown trout population structure and recruitment dynamics

Variation in brown trout (Salmo trutta L.) population recruitment and structure is related to migratory patterns, which should depend on ease of access to habitats providing increased opportunity for growth. We quantified the number of young of year (YOY) as a proportion of the total number of brown trout at 24 locations on 11 streams within the Taieri catchment, New Zealand, including back calculated growth rates and emergence dates from otoliths. Locations with high absolute and relative abundance of YOY fish were related to elevation and distance from the river mainstem (habitat used by migratory fish), fish density, and the interaction between invertebrate food biomass, distance and elevation. Hatch date and growth were not related to the proportion of YOY fish, though growth was negatively correlated to total fish density. We suggest landscape features play a large role in determining recruitment and population structure. Locations at lower elevations have a high YOY density, high competition and lower growth, likely prompting out‐migration. These conditions could be created by successful return migration and spawning of large fecund fish resulting in YOY densities exceeding the habitat carrying capacity. Environmental factors, such as food availability, also played a role in determining population structure. These results provide an example of how population structure and recruitment might be controlled by local conditions and access to high growth environments in wild populations of introduced brown trout across a catchment.