ALBERT

Description: This contribution aims to report the reflections we had with the scientific community during two international workshops on reference materials for stable isotopes in Davos (2002) and Nice (2003). After evaluating the isotopic homogeneity of some existing reference materials, based on either certificates, literature data or specific inter-laboratory rounds, we confirm these as primary reference materials or propose new ones relative to which stable isotope compositions should be reported. We propose DSM-3 for Mg, NIST SRM 915a for Ca, L-SVEC for Li and NBS28 for Si. Cadmium does not yet have a well identified delta zero material, although three commercial mono-elemental Cd solutions have yielded the same isotopic composition relative to one another. In order to scale the linearity of any mass spectrometer, some secondary reference materials are also proposed: Cambridge-1 solution for Mg, the “Münster-Cd” and JEPPIM Cd solutions for Cd and the “Big Batch” silicate for Si. The team from Nancy propose to prepare a mixed spike solution for Li isotopes. Well-characterised natural samples such as ocean or continental waters, diatoms, sponges, rocks and minerals are needed to validate the entire analytical procedure, particularly to take into account the effect of sample mineralisation and of chemical manipulations for elemental separation prior to analysis.

Description: Ecological stoichiometry describes the biochemical constraints of trophic interactions emerging from the different nutrient content and nutrient demand of producers and consumers, respectively. Most research on this topic originates from well-mixed pelagic food webs, whereas the idea has received far less attention in spatially structured habitats. Here, we test how light as well as grazing and nutrient regeneration by consumers affects growth and biomass of benthic primary producers. In the first laboratory experiment, we manipulated grazer presence (two different snail species plus ungrazed control), in the second experiment we factorially combined manipulation of grazer presence and light intensity. We monitored snail and periphyton biomass as well as dissolved and particulate nutrients (nitrogen and phosphorus) over time. Grazers significantly reduced algal biomass in both experiments. Grazers affected periphyton nutrient content depending on the prevailing nutrient limitation and their own body stoichiometry. In the nitrogen (N-) limited first experiment, grazers increased N both in the periphyton and in the water column. The effect was stronger for grazers with lower N-content. In the phosphorus (P-) limited second experiment, grazers increased the P-content of the periphyton, but the grazer with lower N-content had additionally positive effects on algal N. Light reduction did not affect periphyton biomass, but increased chlorophyll-, N- and P-content of the periphyton. These experiments revealed that the indirect effects of grazers on periphyton were bound by stoichiometric constraints of nutrient incorporation and excretion.

Description: Ecosystem resistance to a single stressor relies on tolerant species that can compensate for sensitive competitors and maintain ecosystem processes, such as primary production. We hypothesize that resistance to additional stressors depends increasingly on species tolerances being positively correlated (i.e. positive species co-tolerance). Initial exposure to a stressor combined with positive species co-tolerance should reduce the impacts of other stressors, which we term stress-induced community tolerance. In contrast, negative species co-tolerance is expected to result in additional stressors having pronounced additive or synergistic impacts on biologically impoverished functional groups, which we term stress-induced community sensitivity. Therefore, the sign and strength of the correlation between species sensitivities to multiple stressors must be considered when predicting the impacts of global change on ecosystem functioning as mediated by changes in biodiversity.

Description: Recent experiments, mainly in terrestrial environments, have provided evidence of the functional importance of biodiversity to ecosystem processes and properties. Compared to terrestrial systems, aquatic ecosystems are characterised by greater propagule and material exchange, often steeper physical and chemical gradients, more rapid biological processes and, in marine systems, higher metazoan phylogenetic diversity. These characteristics limit the potential to transfer conclusions derived from terrestrial experiments to aquatic ecosystems whilst at the same time provide opportunities for testing the general validity of hypotheses about effects of biodiversity on ecosystem functioning. Here, we focus on a number of unique features of aquatic experimental systems, propose an expansion to the scope of diversity facets to be considered when assessing the functional consequences of changes in biodiversity and outline a hierarchical classification scheme of ecosystem functions and their corresponding response variables. We then briefly highlight some recent controversial and newly emerging issues relating to biodiversity-ecosystem functioning relationships. Based on lessons learnt from previous experimental and theoretical work, we finally present four novel experimental designs to address largely unresolved questions about biodiversity-ecosystem functioning relationships. These include (1) investigating the effects of non-random species loss through the manipulation of the order and magnitude of such loss using dilution experiments; (2) combining factorial manipulation of diversity in interconnected habitat patches to test the additivity of ecosystem functioning between habitats; (3) disentangling the impact of local processes from the effect of ecosystem openness via factorial manipulation of the rate of recruitment and biodiversity within patches and within an available propagule pool; and (4) addressing how non-random species extinction following sequential exposure to different stressors may affect ecosystem functioning. Implementing these kinds of experimental designs in a variety of systems will, we believe, shift the focus of investigations from a species richness-centred approach to a broader consideration of the multifarious aspects of biodiversity that may well be critical to understanding effects of biodiversity changes on overall ecosystem functioning and to identifying some of the potential underlying mechanisms involved.

Description: Marine and fisheries scientists are increasingly using metapopulation concepts to better understand and model their focal systems. Consequently, they are considering what defines a metapopulation. One perspective on this question emphasizes the importance of extinction probability in local populations. This view probably stems from the focus on extinction in Levins' original metapopulation model, but places unnecessary emphasis on extinction–recolonization dynamics. Metapopulation models with more complex structure than Levins' patch-occupancy model and its variants allow a broader range of population phenomena to be examined, such as changes in population size, age structure and genetic structure. Analyses along these lines are critical in fisheries science, where presence–absence resolution is far too coarse to understand stock dynamics in a meaningful way. These more detailed investigations can, but need not, aim to assess extinction risk or deal with extinction-prone local populations. Therefore, we emphasize the coupling of spatial scales as the defining feature of metapopulations. It is the degree of demographic connectivity that characterizes metapopulations, with the dynamics of local populations strongly dependent upon local demographic processes, but also influenced by a nontrivial element of external replenishment. Therefore, estimating rates of interpopulation exchange must be a research priority. We contrast metapopulations with other spatially structured populations that differ in the degree of local closure of their component populations. We conclude with consideration of the implications of metapopulation structure for spatially explicit management, particularly the design of marine protected area networks.

Description: Three simple fisheries indicators are presented: (i) percentage of mature fish in catch, with 100% as target; (ii) percent of specimens with optimum length in catch, with 100% as target; and (iii) percentage of ‘mega-spawners‘ in catch, with 0% as target, and 30–40% as representative of reasonable stock structure if no upper size limit exists. Application of these indicators to stocks of Gadus morhua, Sardinella aurita and Epinephelus aeneus demonstrate their usefulness. It is argued that such simple indicators have the potential to allow more stakeholders such as fishers, fish dealers, supermarket managers, consumers and politicians to participate in fisheries management and eventually hold and reverse the global pattern of convenience overfishing, which is defined here as deliberate overfishing sanctioned by official bodies who find it more convenient to risk eventual collapse of fish stocks than to risk social and political conflicts.

Description: Conceptual models predict counteractive effects of herbivores and nutrient enrichment on plant diversity and reversed effects of grazers under different nutrient regimes. I tested these hypotheses in 11 field experiments with periphyton communities in three different aquatic habitats (a highly eutrophic lake, an meso-eutrophic lake, and an meso-eutrophic part of the Baltic Sea coast) and in different seasons. Grazer access and nutrient supply were manipulated in a factorial design. Species richness and evenness were chosen as response variables. Both manipulated factors had significant and contrasting effects on diversity, with variable effect strength between sites and seasons. From the two aspects of diversity, evenness well reflected the changes in community composition. Fertilization tended to increase the dominance of few species and thus to decrease evenness, whereas grazers counteracted these effects by removing dominant life forms. The response of species richness was not as expected, since grazers decreased richness throughout, whereas nutrients had weaker effects but tended to increase richness. Species richness rather reflected changes in periphyton architecture. Grazers reduced algal richness presumably by co-consumption of rare species in the tightly connected periphyton assemblages, whereas enrichment may increase richness by providing more structure via increased dominance of filamentous species. Although grazer and nutrient effects on richness and evenness were opposing, there was no change in the effect of one factor by manipulation of the other.

Description: A set of histological characteristics to judge ovarian development was established and used to elaborate morphological criteria of 10 maturity stages of Baltic cod Gadus morhua sampled throughout the annual cycle to represent different macroscopic maturity stages. The applied characteristics confirmed most stages of the macroscopic scale, but the separation of late immature and resting mature females remained imprecise. Atretic vitellogenic oocytes or encapsulated residual eggs identified the resting condition morphologically, but not all ovaries with visible signs of previous spawning showed such features. One ovarian stage that was previously classified as ‘ripening’ was changed to ‘spawning’, owing to the prevalence of hydrated eggs and empty follicles. Ovaries with malfunctions were defined by a separate stage. Macroscopic criteria were revised by comparing the gross anatomy of ovaries with their histology. Female length and gonado-somatic index supported stage definitions, but substantial variation in Fulton's condition factor and the hepato-somatic index rendered these of little use for this purpose. The time of sampling influenced staging accuracy. A female spawner probability function based on the proportion of ripening and ripe specimens in early spring seems to be the most appropriate method to estimate spawner biomass and reproductive potential.

Description: During peak spawning of sprat Sprattus sprattus in the Baltic Sea in May–June egg specific gravity averaged ±s.d. 1·00858 ± 0·00116 g cm−3 but was significantly higher in the beginning and significantly lower towards the end of the spawning season. A close relationship was found between egg diameter and egg specific gravity (r2 = 0·71). This relationship, however, changed during the spawning season indicating that some other factor was involved causing the decrease in specific gravity during the spawning period. The vertical egg distribution changed during the spawning season: eggs were distributed mainly in the deep layers early in the season, occurred in and above the permanent halocline during peak spawning, and above the halocline towards the end of the spawning season. Consequently, poor oxygen conditions in the deep layers and low temperatures in layers between the halocline and the developing thermocline may affect egg development. Thus, opportunities for egg development vary over the spawning season and among spawning areas, and depending on frequency of saline water inflows into the Baltic Sea and severity of winters, between years

Description: A compilation of δ44/40Ca (δ44/40Ca) data sets of different calcium reference materials is presented, based on measurements in three different laboratories (Institute of Geological Sciences, Bern; Centre de Géochimie de la Surface, Strasbourg; GEOMAR, Kiel) to support the establishment of a calcium isotope reference standard. Samples include a series of international and internal Ca reference materials, including NIST SRM 915a, seawater, two calcium carbonates and a CaF2 reference sample. The deviations in δ44/40Ca for selected pairs of reference samples have been defined and are consistent within statistical uncertainties in all three laboratories. Emphasis has been placed on characterising both NIST SRM 915a as an internationally available high purity Ca reference sample and seawater as representative of an important and widely available geological reservoir. The difference between δ44/40Ca of NIST SRM 915a and seawater is defined as -1.88 O.O4%o (δ44/42CaNISTSRM915a/Sw= -0.94 0.07%o). The conversion of values referenced to NIST SRM 915a to seawater can be described by the simplified equation δ44/40CaSa/Sw=δ44/40CaSa/NIST SRM 915a - 1.88 (δ44/42CaSa/Sw=δ44/42CaSa/NIST SRM 915a - 0.94). We propose the use of NIST SRM 915a as general Ca isotope reference standard, with seawater being defined as the major reservoir with respect to oceanographic studies.

Description: Modern models for the development and evolution of the geochemical heterogeneity in the Earth's mantle and the genesis of mantle magmas attach much importance to the processes of interaction between deepseated rocks and metasomatic fluids, which are able, when occurring under mantle conditions, to dissolve significant amounts of major and trace elements (see, for example, [1]). Fluorine is one of the major anions of natural fluids and also one of the principal complex-forming ligands of several metals. To evaluate the possible role of fluorine in the processes of mantle magma genesis and to identify the source of this element in natural magmas in various geodynamic environments, it is necessary to know the fluorine concentration in primitive mantle melts. These data are still relatively scarce, particularly for low alkaline magmas [2-4]. The fluorine concentrations in magmas from suprasubduction zones, whose genesis is largely controlled by the interaction between mantle rocks and fluids, remain poorly known and need further refinement. Data presented in this paper are among the first to characterize the concentrations of fluorine in primitive magmas of suprasubduction zones. These data were obtained by secondary ion mass spectrometry of chill glasses from the lava complex of the Troodos ophiolites in Cyprus. Along with information on the concentrations of major and trace elements, H2O, and Cl in the glasses, our results make it possible to utilize the example of the Troodos ophiolites to characterize the main regularities in the geochemistry of fluorine during the origin of magmas above subduction zones and to assay the contributions of various components that participated in the processes of mantle melting. These data are among the first to demonstrate that subduction-related melts became enriched in F relative to LREE.

Description: The living coelacanth Latimeria chalumnae has a unique position in world biodiversity which raises important questions about conservation and ethics. Some relevant details of coelacanth biology are summarized, including those obtained by direct observation from submersibles. The importance of the coelacanth for evolutionary theory and palaeontology is shown to be paralleled in cultural, literary and artistic areas of human heritage. Threats to the Comoran coelacanths from artisanal fishing are described and conservation measures discussed in relation to local customs and economies as well as the promotion of tourism to spread a new awareness and concern for coelacanths worldwide.

Description: Phylogenetic analyses, using 482 bp of the mitochondrial 16S rDNA and 461 bp of the control region of 16 Diplodus species and Oblada melanura, Pagellus bogaraveo and Pagellus acarne, all close relatives of Diplodus, identified the two representatives of Pagellus as the sister group of Diplodus. Oblada melanura was confirmed as the sister taxon of D. puntazzo, despite its different dental morphology and ecology. Within the genus Diplodus, three clades were identified, the first containing D. annularis and D. bellottii, the second D. vulgaris and D. prayensis, and the third comprising three subclades. These were formed by O. melanura clustering with D. puntazzo, D. fasciatus with D. cervinus, and by the Diplodus sargus sub-species assemblage which also included the West Atlantic taxa D. argenteus, D. bermudensis, D. holbrooki, and the Red Sea endemic D. noct. All members of the D. sargus assemblage were genetically closely related. Among them, D. sargus lineatus from the Cape Verde islands was resolved as most ancestral branch, pointing to the possibility that the diversification and spread of the D. sargus assemblage originated in this region. The hypothesis of stepwise speciation following colonization events within the D. sargus complex is fully supported by phylogenetic reconstruction.

Description: Destructive macroalgal mass blooms threaten estuarine and coastal ecosystems worldwide. We asked which factors regulate macroalgal bloom intensity, distribution and species composition. In field experiments in the Baltic Sea, we analyzed the relative effects of nutrients, herbivores and algal propagule banks on population development and dominance patterns in two co-occurring bloom-forming macroalgae, Enteromorpha intestinalis and Pilayella littoralis. Both species were highly affected by the combined effects of a propagule bank, herbivory and nutrients. The magnitude of effects varied with season. The propagule bank was an important overwintering mechanism for both algae, and allowed for recruitment two months earlier than recruitment via freshly dispersed propagules. This provided a seasonal escape from intense herbivory and nutrient limitation later in the year. Favored by massive recruitment from the propagule bank, Enteromorpha was the superior space occupier in early spring, thereby reducing recruitment of Pilayella. Elimination of the propagule bank and recruitment via freshly dispersed propagules favored Pilayella. Strong and selective herbivory on Enteromorpha supported Pilayella in the presence, but not in the absence of the propagule bank. Nutrient enrichment in summer counteracted herbivore pressure on Enteromorpha, thereby negatively affecting Pilayella. Herbivore and nutrient effects were more pronounced for early life stages than adult algae. These results show that recruitment processes and forces affecting early life stages at the beginning of the vegetation period determine development and dominance patterns of macroalgal blooms. Herbivores naturally suppress blooms but increasing nutrient enrichment can override this important control mechanism. The propagule bank plays a previously unrecognized role for population and community dynamics.

Description: In both terrestrial and aquatic environments introductions of non-indigenous species are continuing and represent one important component of global change. Negative biotic interactions by resident species may prevent successful invaders from becoming pests. Few experimental data are available on the presence and significance of such biotic resistance other than predation or competition. This study addresses the role of habitat structure provided by a native eelgrass (Zostera marina) canopy on growth and survival of the non-indigenous mussel Musculista senhousia, a habitat-modifying gregarious suspension feeder with strong effects on native infauna and eelgrass. In 2 southern California bays, a series of transplantation experiments using tagged mussels revealed that inside an eelgrass canopy, Musculista growth rates were reduced by more than half in 3 of 4 experiments compared to adjacent unvegetated areas. Musculista survival also decreased inside the vegetation in a 4-mo experiment. As one element of habitat structure, we tested the effects of eelgrass patch size, using natural (1 site) and planted (1 site) eelgrass patches of defined sizes. Growth rates of Musculista were highest outside the vegetation and decreased as eelgrass patch size increased. As a potential mechanism for the canopy effects, we suggest that Musculista receives less food inside the vegetation. In the experimental plots, the presence and spatial extent of the macrophyte canopy strongly affected near bottom (10 cm) horizontal water flow assessed with a direct dye tracking method. Reduced mussel growth rates were linearly associated with lower water flow, and presumably, food flux. Over a period of 7 mo, food resources (particulate chlorophyll a) were consistently lower 1 and 5 cm above the sea floor inside eelgrass patches compared to the sand flat. The reduction in food availability matched the growth reduction of Musculista. Also, mussel condition (dry flesh mass/shell mass) was worse in individuals growing in eelgrass than in the sand flat. Previous experiments revealed that dense beds of Musculista impede the rhizome growth and vegetative propagation of eelgrass, yet mussels attain abundances sufficient for interference only if eelgrass beds are patchy. Thus, anthropogenic disturbances on eelgrass beds, which often result in meadow fragmentation, and the proliferation of Musculista may have synergistic negative effects on the persistence of eelgrass beds.

Description: In situ experiments were run with the seastar Asterias rubens to investigate the influence of epibiosis on predation preferences. Mussels (Mytilus edulis) monospecifically fouled by different epibiont species (the barnacle Balanus improvisus, the red filamentous alga Ceramium strictum, the sponge Halichondria panicea and the hydrozoan Laomedea flexuosa) and macroscopically clean mussels were exposed and seastar predation was monitored by SCUBA. Asterias rubens preferred macroscopical unfouled mussels as prey. Fouling generally reduced predation pressure on the mussel hosts (associational resistance). Barnacles protected mussels less efficiently than hydrozoans or algae. We hypothesize that in top-down controlled communities this influence of epibiosis on predation pressure should affect mussel community patterns. A survey of natural mussel-epibiont distribution in the presence or absence of A. rubens showed that the prevalence of differently fouled mussels differed between predation-exposed and predation-protected habitats. Natural mussel-epibiont associations reflected the preferential predation of the major local predators. Additionally, higher epibiotic diversity and evenness could be observed at locations accessible to benthic predators as compared with habitats protected from predation. As blue mussels and seastars are important structuring and controlling elements in the shallow water community of Kiel Fjord, major consequences of epibiosis on the entire system are discussed.

Description: The use of stable isotope analysis in ecological and wildlife studies is rapidly increasing. Studies include evaluating flow of nutrients in ecosystems and studying dietary composition of individual animals. Several mixing models have been developed to evaluate the relative contribution of different foods to the diet of consumers. All these mixing models require that all prey types will be significantly different in bivariate space. This requirement usually poses a problem in analyzing data of stable isotope ratios because sample sizes in most studies are small and seldom normally distributed. We propose a randomization test that we based on the K nearest-neighbor approach. Results from our simulations of power revealed that the K nearest-neighbor test appears to have high power even with small sample sizes and comparatively low displacement. The K nearest-neighbor test described here provides the preliminary statistical analysis necessary for the use of the mixing models, and therefore is a new, powerful tool for analyzing stable isotope data. In evaluating the test performance on data collected from American martens (Martes americana) and their prey on Chichagof Island, Southeast Alaska, we were able to reject our null hypothesis that all samples of prey were drawn from identical populations (P = 0.05). A program written in Pascal or S-Plus is available from the authors to evaluate the K nearest-neighbor statistic for several groups.

Description: Newly hatched Baltic cod Gadus morhua larvae are typically found at depths 〉60 m. This is a region of low light and prey availability, hence generating the hypothesis that larvae have to migrate from hatching depth to the surface layer to avoid starvation and improve their nutritional condition. To test this hypothesis, Baltic cod larvae were sampled during the spawning seasons of 1994 and 1995 with depth-resolving multiple opening/closing nets. Each larva was aged by otolith readings and its RNA/DNA ratio was determined as a measure of nutritional condition. The RNA/DNA ratios of these larvae aged 2-25 days (median 10 days) ranged from 0.4 to 6.2, corresponding to levels exhibited by starving and fast-growing larvae in laboratory calibration studies (starvation, protein growth rate, Gpi= -12.2% day−1; fastgrowing larvae, Gpi=14.1%day−1) respectively. Seventy per cent of the field caught larvae had RNA/DNA ratios between the mean values found for starving and fed laboratory larvae. Only larvae aged 8-11 days had higher mean RNA/DNA ratios above 45 m than below (t-test, P〈0.05). However, the instantaneous protein growth rates were significantly higher for all larval age groups in the surface layers (t-test, P〈0.05). Starving larvae were found in all depths sampled (10-85 m), whereas growing larvae (positive Gpi) were restricted to samples taken shallower than 45 m. These superior growth rates above 45 m corroborate the hypothesis and imply that migration to the shallow water layers is a prerequisite for good nutritional condition, growth and survival of Baltic cod larvae. The frequent occurrence of cod larvae older than 8 days in the deep water in poor condition suggests that a proportion of the larvae will die from Starvation in the deep layers of the Baltic Sea.

Description: We propose that ocean conditions of the Near Islands in the western Atleutian Arc mimic those of the shallow continental shelf of the eastern Bering Sea to the extent that the marine community, including assemblages of forage fishes and their avian predators, has disinctly coastal characteristics. In contrast, marine avifauna and their prey at neighbouring Buldir Island are distinctly oceanic. For example, at the Near Islands, the ratio of thick-billed to common murres, Uria lomvia and U. aalge, is low and black.legged kittiwakes, Rissa tridacytla, but not red-legged kittiwakes, R. brevirostris, nest there. Diets of murres and kittiwkaes are dominated by sand lance, Ammodytes hexapterus, an abundant coastal species. At Buldir Island, thick-billed murres greatly outnumber common murres, red-legged kittiwakes and black-legged kittiwakes are both abundant, and diets of the birds consist primarily of oceanic squid and lantern-fish (Myctophidae). This mesoscale difference in food webs is apparently a consequence of the local physiography. A broad escarpment on the Near physiographic block creates a comparatively expansive, shallow, shelf-like habitat around the Near Islands, where a pelagic community typical of coastal regions flourished. Buldir Island is the only emergent feature of the Buldir physiographic block, with little shallow water surrounding it and, apparently, little opportunity for other than oceanic species to exist. Patterns in the distribution of fishes, and thus of sea birds, throughout the Atleutian Islands might be largely explained by the relationship between physical environments and food webs. In the larger context of fisheries oceanography, this model for the Aleutian Islands improves our ability to interpret physical and biological heterogeneity in the ocean and its relationship to regional community dynamics and trends in the abundance and productivity of individual species at higher tropic levels.

Description: The temperature relationship of routine metabolic rate (Rr) of non-feeding, non-growing Coregonus lavaretus larvae between 2 and 15°C is characterized by Q10-values ranging from l.8-2.45. The rate of growth, based on weight determinations, of first-feeding larvae amounted to 3.5, 7.6 and 9.4% day-1 at 5, 10 and 12°C respectively, from which Q10-values between 4.0 and 4.8 can be calculated. The rate of increase of muscle mass between 5 and 10°C, based on the determination of the cross-sectional area of inner muscle fibres, resulted in a Q10-value of 4.5. Water temperature influenced the pattern of growth of the inner muscle fibres. At hatching, after 360 day degrees, total muscle mass of larvae reared at 4 and 8°C was independent of temperature, but at 4°C the rate of mass increase owed more to hyperplasia (increase in fibre number) than to hypertrophy (increase in fibre mass), whereas at 8°C the opposite was the case. The calculation of power budgets (including the metabolic cost of growth) of first-feeding larvae yielded net conversion efficiencies (K2) increasing with temperature from 46.3% at 5°C to 54.7% at 12°C. Comparing our data with literature data two general conclusions can be drawn. (1) In first-feeding larvae the net, but not the gross, conversion efficiency of food energy increases with temperature. This is due to net energy input being characterized by a much higher Q10-value than energy expenditures. (2) In embryos of freshwater fish so far investigated hyperplasia plays a greater role in the increase of fibre mass than hypertrophy at the lower temperature, whereas in embryos of marine fish hyperplasia prevails at the higher temperature. It is suggested that this discrepancy correlates with the high concentration of free amino acids in the eggs of marine species which provide an additional, easily available, source of metabolic energy absent in freshwater species.