Estimating salinity stress via hsp70 expression in the invasive round goby (Neogobius melanostomus): implications for further range expansion

Species invasions often occur on coasts and estuaries where abiotic conditions vary, e.g. salinity, temperature, runoff etc. Successful establishment and dispersal of non-indigenous species in many such systems are poorly understood, partially since the species tend to show genetic and ecological plasticity at population level towards many abiotic conditions, including salinity tolerance. Plasticity may be driven by shifting expression of heat shock proteins such as Hsp70, which is widely recognized as indicator of physical stress. In this study, we developed a qPCR assay for expression of the hsp70 gene in the invasive round goby (Neogobius melanostomus) and tested the expression response of fish collected from a brackish environment in the western Baltic Sea to three different salinities, 0, 10 and 30. hsp70 expression was highest in fresh water, indicating higher stress, and lower at brackish (ambient condition for the sampled population) and oceanic salinities, suggestive of low stress response to salinities above the population’s current distribution. The highest stress in fresh water was surprising since populations in fresh water exist, e.g. large European rivers and Laurentian Great Lakes. The results have implications to predictions for the species’ plasticity potential and possible range expansion of the species into other salinity regimes.

     

The osmoregulatory ability of the invasive species sunbleak Leucaspius delineatus and topmouth gudgeon Pseudorasbora parva at elevated salinities, and their likely dispersal via brackish waters

The European invasive species sunbleak Leucaspius delineatus and topmouth gudgeon Pseudorasbora parva , previously thought to be stenohaline freshwater fishes, were shown to tolerate salinities of 10·8 and 13·7 for up to 4 days. The increased plasma osmolality and ion concentrations, and decreased body water content, however, indicated that they are unable to hypo-osmoregulate. Therefore there is limited potential for these species to disperse into new river systems via brackish waters with a salinity >13·7.     

Interaction of salinity and temperature as a mechanism for spatial separation of three co-existing species of Ambassidae (Cuvier) (Teleostei) in estuaries on the south-east coast of Africa

Ambassidae are represented in South African estuaries by three species which are very similar in external morphology, feeding ecology, alimentary system and distribution. The co-existence and spatial separation of these species of Ambassis in the estuaries of southern Africa can be explained by the tolerance of each to salinity and temperature. Investigations of the temperature tolerance ranges of the three species suggest that the osmoregulatory capability of Ambassis productus in reduced salinities (<10%) increases while that of A. gymnocephalus decreases sharply in salinities below 20%. A. natalensis , which is endemic to the south-east coast of Africa, is adapted to a wide range of estuarine conditions. Interaction between salinity and temperature tolerances on the limits within which Ambassis spp. survive is significant with regard to the distribution of the three species in estuaries. Catch data showed that A. productus is restricted to areas of low salinity and seasonal low temperature (upper reaches of estuaries), A. gynmocephalus to high salinities >25% (estuary mouths) and A. natalensis to intermediate salinities associated with the middle reaches of estuaries.     

Comparison of low salinity tolerance in Callinectes sapidus Rathbun and Callinectes similis Williams postlarvae upon entry into an estuary

Planktonic larvae of estuarine crabs are commonly exported to the continental shelf for development and then return to coastal and estuarine areas as postlarvae (megalopae). Megalopae returning to estuaries must be adapted to survive in brackish water whereas those of coastally distributed species should not need such adaptations. We investigated 1) whether megalopae of the estuarine crab Callinectes sapidus and the coastal crab Callinectes similis undergo changes in salinity tolerance upon entry into an estuary and 2) what factors induce those changes. Megalopae were collected at a coastal site and a nearby estuarine site and exposed to a range of salinities (5, 10, 15, 20 and 30) for 6 h. Percent survival was determined after 24 h reintroduction to the collection site water. We also investigated 1) whether increased salinity tolerance was induced by reduced salinity or estuarine chemical cues, 2) the time to acclimation and 3) the salinity necessary for acclimation. C. sapidus megalopae from the estuarine site were more likely to survive exposure to low salinities than those from the coastal site. C. sapidus megalopae from the coastal site exhibited increased survival after acclimation to salinities of 27 and 23 for 12 h. Estuarine chemical cues had no effect on salinity tolerance. C. similis megalopae were less likely to survive at low salinities and did not exhibit an acclimation response upon exposure to reduced salinities. These results suggest that megalopae of C. sapidus are physiologically adapted to recruit to estuaries whereas megalopae of C. similis are unable to acclimate to low salinity conditions.     

Effects of salinity on the growth and morphology of the invasive,euryhaline hydroid Cordylophora (Phylum Cnidaria,Class Hydrozoa)

The invasive, euryhaline hydroid Cordylophora sp. is a colonial cnidarian present in both freshwater and brackish water habitats. Individuals contend with osmotic stress at the tissue and cellular level. It has been suggested that this hydroid's ability to expand its range of distribution by invading new habitats is due in large part to an ability to acclimate to new salinities. The purpose of this study was to assess colony growth and morphological changes at various salinities in freshwater and brackish genotypes of Cordylophora sp. Single genotypes from a known freshwater clade (0.5 psu; Des Plaines River) and a known brackish clade (16 psu; Napa River) were cultured and gradually transitioned to 12 different salinities ranging 0.5–22 psu, and we characterized the growth rates and hydranth morphological features at each salinity. Colony growth was optimal at 0.5 psu for the freshwater genotype and 10 psu for the brackish genotype. Changes in hydranth morphology in the freshwater genotype were primarily observed at higher salinities, while morphological changes in the brackish genotype primarily occurred at lower salinities. Our results for the brackish genotype generally concur with previous work, but this study is the first to document the response of a freshwater genotype of Cordylophora sp. to various salinities. Differences in growth between these two genotypes strongly support the previously proposed existence of multiple cryptic species. Furthermore, because this hydroid is quite prevalent in freshwater and brackish systems as a fouling organism, understanding the effects of various salinities on the successful establishment of Cordylophora sp. is an important contribution to the understanding of the ecophysiology and management of this invasive hydroid.     

Species richness of crustacean zooplankton and trophic structure of brackish lagoons in contrasting climate zones: north temperate Denmark and Mediterranean Catalonia (Spain)

We sought to identify environmental factors influencing crustacean zooplankton species richness in brackish lagoons and to elucidate whether crustacean zooplankton species richness and trophic structure of brackish lagoons differ among two regions with contrasting temperatures. We sampled 35 and 42 brackish lagoons (salinity ranging from 0.3 to 55‰) in Mediterranean Catalonia (NE Spain) and northern-temperate Denmark, respectively. No significant differences were found in total crustacean zooplankton species richness or cladoceran richness between the climatic regions. Calanoid richness was higher in Denmark than in Catalonia, while cyclopoid richness was higher in Catalonia. Salinity was the most important variable associated with zooplankton species richness in both regions, richness of total zooplankton species, cladocerans and cyclopoids being negatively related with salinity. In both regions, a shift occurred from dominance of large filter feeding cladoceran species at low salinities to copepods and small cladoceran species at higher salinities. Cladoceran richness increased with increasing total phosphorus, but was not influenced by total nitrogen or chlorophyll-a. Trophic structure in Mediterranean brackish lagoons showed a more pronounced seasonal variation than in north temperate brackish lagoons. Our results imply that the indirect effects of climate warming, such as changes in salinity and hydrology, will have a larger impact on brackish lagoon ecosystems than the increase in temperature per se.     

Novel distribution pattern between coexisting sexual and obligate asexual variants of the true estuarine macroalga Ulva prolifera

Where sexual and asexual forms coexist within a species, the asexuals are often found to be prevalent in marginal habitats. This asexual distribution pattern has received evolutionary attention linked to the paradox of sex. In many marine species, there is a distributional trend of asexual modes being more common in lower salinity waters regarded as the ecogeographic marginal, being explained by negative effects of low salinities on sexual reproductive success. However, the distribution pattern of estuarine species recently adapted to low salinity waters has remained unknown. The brackish macroalga Ulva prolifera being a major benthic component of estuarine ecosystems includes a sexual variant and obligate asexual variants by means of motile spores. We examined the sexual–asexual distribution pattern of this alga along a salinity gradient in river estuaries. Surprisingly, opposite to the distributional trend of marine organisms, the results clearly showed the persistent predominance of sexuals in the lower salinity reaches than the asexuals. In addition, a frequent alternating of dioecious gametophytes and sporophytes in the sexual population was observed, suggesting the sexual reproductive process would be robustly performed in the low salinity waters. Considering U. prolifera had evolved from the ancestral marine species to become a true estuarine species of which the core habitat is the low salinity reaches, in a broad sense its sexual–asexual distribution pattern would be involved in asexual marginal occupations of the species range previously reported in other organisms. Based on the frozen niche variation model, we can give a concise explanation for the evolutionary process of this pattern; multiple asexuals with frozen genotypic variation had arisen from sexual ancestors undergoing low salinity adaptation and share the estuarine habitat with the sexuals at present.     

Laboratory simulation of the effects of environmental salinity on wild‐caught juveniles of European sea bass Dicentrarchus labrax and gilthead seabream Sparus aurata

Gilthead seabream Sparus aurata and European sea bass Dicentrarchus labrax , are two important species in Mediterranean aquaculture. In the wild, their juveniles occur in brackish areas such as lagoons and river deltas. Even though the juveniles seem to favour brackish environments, low salinity incurs an energy cost for osmoregulation. This paper presents the results of a series of laboratory experiments exploring the effects of salinity on growth, feeding, food conversion, survival and maintenance energy requirements of wild‐caught juveniles. The fish were kept in the laboratory, divided in groups of 20 in small tanks of 50 l each, and supplied with biologically filtered seawater of four salinity levels (8, 18, 28‰ and natural seawater) and fixed temperature (20 ± 1·4° C). The fish were fed pelleted feed throughout the experiment. Both species showed great similarity in their responses to lower salinities. Satiation time for both species increased with decreasing salinity, while maintenance requirements (required daily ration and energy) increased as with increasing salinity. Growth and feed conversion is highest for salinities around 28‰ and lower for salinities above and below. Both species share common physiological features, and intermediate salinities are optimal for their performance in nature and in captivity.     

A fundamental difference between macrobiota and microbial eukaryotes: protistan plankton has a species maximum in the freshwater-marine transition zone of the Baltic Sea

Remane's Artenminimum at the horohalinicum is a fundamental concept in ecology to describe and explain the distribution of organisms along salinity gradients. However, a recent metadata analysis challenged this concept for protists, proposing a species maximum in brackish waters. Due to data bias, this literature-based investigation was highly discussed. Reliable data verifying or rejecting the species minimum for protists in brackish waters were critically lacking. Here, we sampled a pronounced salinity gradient along a west–east transect in the Baltic Sea and analysed protistan plankton communities using high-throughput eDNA metabarcoding. A strong salinity barrier at the upper limit of the horohalinicum and 10 psu appeared to select for significant shifts in protistan community structures, with dinoflagellates being dominant at lower salinities, and dictyochophytes and diatoms being keyplayers at higher salinities. Also in vertical water column gradients in deeper basins (Kiel Bight, Arkona and Bornholm Basin) appeared salinity as significant environmental determinant influencing alpha- and beta-diversity patterns. Importantly, alpha-diversity indices revealed species maxima in brackish waters, that is, indeed contrasting Remane's Artenminimum concept. Statistical analyses confirmed salinity as the major driving force for protistan community structuring with high significance. This suggests that macrobiota and microbial eukaryotes follow fundamentally different rules regarding diversity patterns in the transition zone from freshwater to marine waters.     

The effects of salinity and temperature on the development and survival of fish parasites

In brackish water the variety of marine and freshwater parasite species is considerably reduced. The distribution in brackish water of most marine endoparasites is restricted by the salinity tolerance of their hosts, most of the parasite species are more tolerant than their hosts. The influence of salinity and temperature on nine species has been examined; first stage larvae of Contracaecum aduncum develop in 0-32‰ salinity; Cryptocotyle lingua proved to be infective at salinities down to 4‰. The greatest resistance was found in Anisakis larvae from herring Clupea harengus , which survived for more than half a year. Parasites in the fish intestines appear to be unaffected by changing water salinities, as the osmolarity in the intestines stays nearly constant. Marine ectoparasites ( Acanthochondria depressa, Lepeophtheirus pectoralis ) survive about three times longer than freshwater species ( Piscicola geometra, Argulus foliaceus ) when salinity is 16‰. High temperature increases the effects of adverse salinities on parasites. There is evidence that none of these ecto-parasitic species can develop within the range of 7-20‰ salinity.     

Comparison of inshore and offshore fish larval assemblages within the wider Yangtze River estuary (China)

It is hypothesized that fish larvae undertake vertical movements in estuarine waters. To test this hypothesis, a sampling period was selected during times when many spring‐spawning fish reach maximum abundance in coastal waters so that spring and summer variations in larval fish assemblages could be determined in the Yangtze River estuary. Six oceanographic surveys were conducted across the salinity gradient of an inshore (freshwater) and offshore area (brackish intersection) during spring (May) and summer (August) between 2010 and 2012. The fish larval community was dominated by species of Engraulidae, Gobiidae, Champsodontidae and Mugilidae. The pre‐flexion and flexion larval stages of euryhaline marine species, which are dependent on estuaries as nursery areas, were common. The brackish and marine larval assemblage was the most abundant with taxa such as Coilia mystus and Engraulis japonicus accounting for more than 57.3% of the total catch. Spatial differences in the taxonomic composition of larval fish assemblages were evident between the inshore and offshore areas. Additionally, the Yangtze River runoff regulatory functions as affected by the Three Gorges Reservoir operational mode (hydrological alternating operations) showed weak influences on fish and habitat environments. Low salinities from high freshwater inflow limited bay anchovy production in the inshore area.     

Different reproductive success at low salinity determines the estuarine distribution of two Palaemon prawn species

The two prawn species Palaemon adspersus Rathke and P. elegans Rathke differ in their distribution patterns in estuaries: P. adspersus occurs at lower salinities and also extends further into the Baltic than P. elegans . Yet, at low salinities adult survival does not differ between the two species. Reproductive success was, however, substantially reduced in P. elegans at low salinity, but not in P. adspersus . Berried P. elegans females from the Swedish west coast hatched significantly fewer clutches at 10%‰ than did P. adspersus females from the same locality. Furthermore, larval survival in P. elegans was significantly lower at 5 and 7.5‰ than in P. adspersus . At higher salinities (10 and 24.5‰) no interspecific differences in larval survival were found, except in one experiment where P. elegans larvae had a lower mortality. It is concluded that the different estuarine distributions of the two palaemonid prawn species result from these interspecific differences in reproductive success at low salinity.     

Influences of Salinity on Growth and Survival of Juvenile Pinfish <Emphasis Type="Italic">Lagodon rhomboides</Emphasis> (Linnaeus)

We investigated the effect of salinity on growth, survival, and condition of pinfish Lagodon rhomboides juveniles (36–80 mm standard length) in two laboratory experiments in July 2003 and June/July 2004. Our results show that juvenile pinfish grown in laboratory conditions under a range of salinities experience rapid growth and high survival in typical estuarine-like salinities (15–30 ppt). We also found that relative weight as an index of condition corroborates the idea that pinfish are well adapted to survive and grow in a wide range of salinities. Such rapid growth and high survival in a dynamic environment may afford juvenile pinfish potential ecological advantages over other estuarine-dependent fish species that are relatively more constrained by changes in salinity regime. Because coastal development is wide-spread throughout Gulf of Mexico and Atlantic estuaries, insights concerning the impacts of human-induced changes to estuarine environments are essential for effective management practices.     

Salinity mediates the competitive interactions between invasive mosquitofish and an endangered fish

The interplay of abiotic factors and competition has a long history in ecology, although there are very few studies on the interaction of salinity and competition in fish. Mosquitofish (Gambusia holbrooki) are among the most invasive fish worldwide, with well documented ecological impacts on several taxa such as amphibians and small native fish. It has been previously hypothesized, based on field observations, that salinity limits the invasive success of mosquitofish and provides a competitive refuge for Mediterranean cyprinodonts. We experimentally tested this hypothesis by examining the agonistic behaviour and food competition between mosquitofish and an endangered native cyprinodont (Aphanius fasciatus) at three salinities (0, 15, 25‰). Intraspecific aggressive behaviour for both species was not significantly affected by salinity. As salinity increased, mosquitofish decreased their aggressive behaviour towards cyprinodonts and captured less prey. In contrast, the cyprinodonts did not change their behaviour with different salinity treatments, with the possible exception of increased defensive acts in higher salinities, but captured more prey with increasing salinity because of the reduced efficiency of mosquitofish. Our study confirms previous field observations that salinity limits the invasive success of mosquitofish and provides one of the few experimental demonstrations that it may mediate behavioural and competitive interactions between fish species. Condition-specific competition of mosquitofish might be expected with other species and ecosystems worldwide and illustrates the importance of integrating biotic and abiotic factors in the study of interspecific interactions.     

Effects of salinity, temperature and individual variability on the reproduction of Eurytemora affinis (Copepoda; Calanoida) from the Seine estuary: A laboratory study

The brackish water copepod Eurytemora affinis is the most abundant copepod species in the low salinity zone (2-15) of the Seine estuary. Despite its ecological importance, little is known about its population dynamics in the Seine. We studied the effects of temperature (10 °C and 15 °C) and salinity (5, 15 and 25) on reproduction under non-limiting food conditions. We used experiments to determine multiple reproductive parameters for E. affinis. In all experiments, we fed E. affinis a mixture of Rhodomonas marina and Isochrysis galbana. Couples of pre-adult females (C5) and adult males were mated until the female extruded a clutch of eggs and then individual females were observed every 6-12 hours until death to determine (a) embryonic development time, (b) inter clutch time and (c) clutch size throughout their adult lifespan. All reproductive parameters were negatively affected by low temperature (10 °C) and by high salinity (25). At 10 °C and a salinity of 25, mortality during the post-embryonic period was extremely high (85%). Differences in all reproductive parameters between salinities 5 and 15 were minimal. From 15 °C to 10 °C mean latency time (time between hatching of eggs and extrusion of new ones) increased from 0.8 to 2.25 days, the mean embryonic development time from 2.2 to 3.2 days and the mean clutch size decreased from 38 to 22 eggs female^- 1. The mean clutch size decreased when females reached a critical age. The hatching success was high (near 95%) under all conditions except at high salinity. Egg production rates showed no significant differences between salinities 5 and 15 and were significantly higher at 15 °C (13 eggs female^- 1 day^- 1 at salinity 5 and 15) than at 10 °C (4 eggs female^- 1 day^- 1). These values at 15 °C were higher compared to those from other populations of E. affinis in estuaries or lakes. The high reproductive potential of E. affinis from the Seine estuary at 15 °C and low salinities explain its high densities in the low salinity zone during spring and early summer.     

Seagrass Proliferation Precedes Mortality during Hypo-Salinity Events: A Stress-Induced Morphometric Response

Halophytes, such as seagrasses, predominantly form habitats in coastal and estuarine areas. These habitats can be seasonally exposed to hypo-salinity events during watershed runoff exposing them to dramatic salinity shifts and osmotic shock. The manifestation of this osmotic shock on seagrass morphology and phenology was tested in three Indo-Pacific seagrass species, Halophila ovalis, Halodule uninervis and Zostera muelleri, to hypo-salinity ranging from 3 to 36 PSU at 3 PSU increments for 10 weeks. All three species had broad salinity tolerance but demonstrated a moderate hypo-salinity stress response – analogous to a stress induced morphometric response (SIMR). Shoot proliferation occurred at salinities <30 PSU, with the largest increases, up to 400% increase in shoot density, occurring at the sub-lethal salinities <15 PSU, with the specific salinity associated with peak shoot density being variable among species. Resources were not diverted away from leaf growth or shoot development to support the new shoot production. However, at sub-lethal salinities where shoots proliferated, flowering was severely reduced for H. ovalis, the only species to flower during this experiment, demonstrating a diversion of resources away from sexual reproduction to support the investment in new shoots. This SIMR response preceded mortality, which occurred at 3 PSU for H. ovalis and 6 PSU for H. uninervis, while complete mortality was not reached for Z. muelleri. This is the first study to identify a SIMR in seagrasses, being detectable due to the fine resolution of salinity treatments tested. The detection of SIMR demonstrates the need for caution in interpreting in-situ changes in shoot density as shoot proliferation could be interpreted as a healthy or positive plant response to environmental conditions, when in fact it could signal pre-mortality stress.     

Genotype-by-environment interaction for salinity tolerance in the freshwater-invading copepod Eurytemora affinis

This study examined the extent of phenotypic plasticity for salinity tolerance and genetic variation in plasticity in the invasive copepod Eurytemora affinis. Euryemora affinis is a species complex inhabiting brackish to hypersaline environments but has invaded freshwater lakes and reservoirs within the past century. Reaction norm experiments were performed on a relatively euryhaline population collected from a brackish lake with fluctuating salinity. Life history traits (hatching rate, survival, and development time) were measured for 20 full-sib clutches that were split and reared at four salinities (fresh, 5, 10, and 27 practical salinity units [PSU]). On average, higher salinities (10 and 27 PSU) were more favorable for larval growth, yielding greater survival and faster development rate. Clutches differed significantly in their response to salinity, with a significant genotype-by-environment interaction for development time. In addition, genetic (clutch) effects were evident in response to low salinity, given that survival in fresh (lake) water was significantly positively correlated with survival at 5 PSU for individual clutches. Clutches raised in fresh water could not survive beyond metamorphosis, suggesting that acclimation to fresh water could not occur in a single generation. Results suggest the importance of natural selection during freshwater invasion events, given the inability of plasticity to generate a freshwater phenotype, and the presence of genetic variation for plasticity upon which natural selection could act.     

Effect of salinity on the distribution of Ponto-Caspian gammarids in a non-native area – environmental and experimental study

The native area of gammarids from the so-called ‘Caspian complex’, Pontogammarus robustoides (G.O. Sars, 1894), Obesogammarus crassus (G.O. Sars, 1894), Dikerogammarus haemobaphes (Eichwald, 1841) and D. villosus (Sowinsky, 1894), is associated with brackish waters. Over the last several decades they have colonized the European inland waters and part of the brackish Baltic Sea. It is believed that anthropogenic increase in the salinity of inland waters facilitated their expansion. However, the influence of salinity on the dispersal of gammarid species outside their native area is not fully understood. We tested the hypothesis that salinity was a major factor in determining distribution, based on the abundance of Gammaridae in three coastal areas of low salinity (brackish Baltic), i.e. 0.3, 3.4 and 7.3 PSU, successfully inhabited by them. Additionally, for the first time, the effect of water salinity on the osmoregulatory capacity of O. crassus was examined under laboratory conditions, for the salinities given above. The experiments showed that similarly as in the case of other Caspian complex species, salinity values of about 7 PSU create better conditions for osmoregulation in O. crassus than lower salinities (i.e. 0.3 and 3.4 PSU). In the environmental part of the study, we observed that only D. villosus achieved a significantly higher abundance in the area of 7.3 PSU. Thus, we concluded that in the range of 0.3–7.3 PSU, salinity is not a key factor governing the distribution of Ponto-Caspian gammarids.     

Impact of prairie fragment size on proportion of females and reproductive success of Lobelia spicata Lam., a gynodioecious species

• Due to ongoing human impacts, plant species increasingly occur in landscapes that are highly fragmented, with remaining natural habitats occupying small areas, resulting in populations that are smaller and more isolated than in previous time periods. This changed metapopulation structure is expected to have negative impacts on seed production. For example, the proportion of female plants within gynodioecious populations may be more volatile due to genetic drift in small populations associated with small habitat fragments, with concomitant impacts on seed production. My aims were to determine: (i) if variation in proportion of females is larger in smaller fragments; and (ii) if such changes in female frequency in small fragments result in reduced seed production.
• Thirty‐two populations of Lobelia spicata Lam., a gynodioecious species, were surveyed in 2000, 2001 and 2009 in the tallgrass prairie region of Midwestern North America (Illinois and Indiana, USA). Data were collected for: proportion of female plants, total number of flowering plants (measure of population size), seed set per plant and prairie fragment size (another measure of population size).
• The proportion of females is more variable in smaller prairie fragments. Seed number per fruit decreases as the proportion of females increases in a population, but only significantly for female plants. The number of flowering plants is positively associated with fruit production for both genders. Populations within larger prairie fragments have higher seed production.
• The reproductive consequences of habitat fragmentation depend on the plant breeding system. While both sexes were negatively impacted, females were more adversely affected.

     

Multiple Euryhaline Lineages of Centrohelids (Haptista: Centroplasthelida) in Inland Saline Waters Revealed with Metabarcoding

The diversity of centrohelids in inland saline waters was studied with metabarcoding for the first time. The fragment of V6–V7 regions of 18S rDNA was sequenced with newly designed primers. Obtained OTUs were identified with molecular phylogenetic analysis and comparison of the signatures in 39es9 hairpin of V7. The obtained data included some OTUs, which could be attributed to four described species, but the majority belonged to previously established or novel environmental clades. Along with some presumably marine/brackish clades and freshwater/low salinity (0–2 ppt) clades, seven presumable species demonstrating broad (from 1–2 up to 78 ppt) salinity tolerance were detected. A number of OTUs belonged to Raphidocystis contractilis, which is known from three independent findings in brackish habitats only. Thus, it was assumed that this species is stenohaline and specifically adapted to salinity 5–15 ppt. The high level of salinity tolerance was suggested for centrohelids before based on morphology, which was used to justify their cosmopolitan distribution. Later these views were criticized based on environmental sequencing, but the results of the current survey indicate, that at least some species are present at salinities from almost freshwater (1–2 ppt) to twice oceanic (78 ppt) and are presumably capable of overcoming oceanic salinity barriers for their distribution.