Aerosolization flux, bio-products, and dispersal capacities in the freshwater microalga Limnomonas gaiensis (Chlorophyceae).

Little is known on the spreading capacities of Limnomonas gaiensis across freshwater lakes in Northern Europe. In this study, we show that the species could successfully be aerosolized from water sources by bubble bursting (2-40 particles.cm-3), irrespectively of its density in the water source or of the jet velocity used to simulate wave breaking. The species viability was impacted by both water turbulences and aerosolization. The survival rate of emitted cells was low, strain-specific, and differently impacted by bubble busting processes. The entity "microalga and bionts" could produce ethanol, and actively nucleate ice (principally ≤-18 °C) mediated soluble ice nucleation active proteins, thereby potentially impacting smog and cloud formation. Moreover, smallest strains could better cope with applied stressors. Survival to short-term exposure to temperatures down to -21 °C and freezing events further suggest that L. gaiensis could be air dispersed and contribute to their deposition.

Physiological responses to pH in the freshwater microalga Limnomonas gaiensis.

The ecological niche of the recently described limnic microalga Limnomonas gaiensis (Chlamydomonadales) in Northern Europe remains unknown. To decipher the species tolerance capacity to pH, the effects of hydrogen ions on the physiological response of L. gaiensis were investigated. Results showed that L. gaiensis could tolerate exposure from pH 3 up to pH 11, with an optimal survival at pH 5-8. Its physiological response to pH was strain specific. Globally the southernmost strain was more alkaliphilic, had a slightly rounder shape, a slowest growth rate, and a lowest carrying capacity. Despite strain discrepancies among lakes, Swedish strains exhibited similar growth rates, faster at more acidic conditions. The extreme pH conditions affected its morphological features such as the eye spot and papilla shape, especially at acidic pH, and the cell wall integrity, at more alkaline pH. The wide range tolerance of L. gaiensis to pH would not be a hindrance to its dispersal in Swedish lakes (pH 4-8). Notably, the storage of high-energetic reserves over a wide range of pH conditions, as numerous starch grains and oil droplets, makes L. gaiensis a good candidate for bioethanol/fuel industrial production and a key resource to sustain aquatic food chain and microbial loop.

Population connectivity, dispersal, and swimming behavior in Daphnia.

The water flea Daphnia has the capacity to respond rapidly to environmental stressors, to disperse over large geographical scales, and to preserve its genetic material by forming egg banks in the sediment. Spatial and temporal distributions of D. magna have been extensively studied over the last decades using behavioral or genetic tools, although the correlation between the two has rarely been the focus. In the present study, we therefore investigated the population genetic structure and behavioral response to a lethal threat, ultraviolet radiation (UVR), among individuals from two different water bodies. Our results show two genetic populations with moderate gene flow, highly correlated with geographical location and with inheritable traits through generations. However, despite the strong genetic differences between populations, we show homogeneous refuge demand between populations when exposed to the lethal threat solar UVR.

Diverging responses to threats across generations in zooplankton.

Our understanding on how organisms evolutionarily cope with simultaneously occurring, multiple threats over generations is still elusive. In a long-term experimental study, we therefore exposed clones of a freshwater cladoceran, Daphnia magna, to threats from predation and ultraviolet radiation (UVR) during three consecutive parthenogenetic generations. We show that Daphnia can adapt to different sets of threats within three generations through modifying morphology, swimming behavior, or life-history traits. When faced with predator cues, D. magna responded with reduced body size, whereas exposure to UVR induced behavioral tolerance when again exposed to this threat. Such UVR-tolerant behavior was initially associated with a reduced clutch size, but Daphnia restored the reproductive output gradually through generations. The findings advance our understanding on how those common invertebrates, with a global distribution, are able to persist and rapidly become successful in a changing environment.

Ice Nucleation Activity and Aeolian Dispersal Success in Airborne and Aquatic Microalgae.

Microalgae are common members of the atmospheric microbial assemblages. Diverse airborne microorganisms are known to produce ice nucleation active (INA) compounds, which catalyze cloud and rain formation, and thus alter cloud properties and their own deposition patterns. While the role of INA bacteria and fungi in atmospheric processes receives considerable attention, the numerical abundance and the capacity for ice nucleation in atmospheric microalgae are understudied. We isolated 81 strains of airborne microalgae from snow samples and determined their taxonomy by sequencing their ITS markers, 18S rRNA genes or 23S rRNA genes. We studied ice nucleation activity of airborne isolates, using droplet freezing assays, and their ability to withstand freezing. For comparison, we investigated 32 strains of microalgae from a culture collection, which were isolated from polar and temperate aqueous habitats. We show that ∼17% of airborne isolates, which belonged to taxa Trebouxiphyceae, Chlorophyceae and Stramenopiles, were INA. A large fraction of INA strains (over 40%) had ice nucleation activity at temperatures ≥-6°C. We found that 50% of aquatic microalgae were INA, but the majority were active at temperatures <-12°C. Most INA compounds produced by microalgae were proteinaceous and associated with the cells. While there were no deleterious effects of freezing on the viability of airborne microalgae, some of the aquatic strains were killed by freezing. In addition, the effect of desiccation was investigated for the aquatic strains and was found to constitute a limiting factor for their atmospheric dispersal. In conclusion, airborne microalgae possess adaptations to atmospheric dispersal, in contrast to microalgae isolated from aquatic habitats. We found that widespread taxa of both airborne and aquatic microalgae were INA at warm, sub-zero temperatures (>-15°C) and may thus participate in cloud and precipitation formation.

The potential for dispersal of microalgal resting cysts by migratory birds.

Most microalgal species are geographically widespread, but little is known about how they are dispersed. One potential mechanism for long-distance dispersal is through birds, which may transport cells internally (endozoochory) and deposit them during, or in-between, their migratory stopovers. We hypothesize that dinoflagellates, in particular resting stages, can tolerate bird digestion; that bird temperature, acidity, and retention time negatively affect dinoflagellate viability; and that recovered cysts can germinate after passage through the birds' gut, contributing to species-specific dispersal of the dinoflagellates across scales. Tolerance of two dinoflagellate species (Peridiniopsis borgei, a warm-water species and Apocalathium malmogiense, a cold-water species) to Mallard gut passage was investigated using in vitro experiments simulating the gizzard and caeca conditions. The effect of in vitro digestion and retention time on cell integrity, cell viability, and germination capacity of the dinoflagellate species was examined targeting both their vegetative and resting stages. Resting stages (cysts) of both species were able to survive simulated bird gut passage, even if their survival rate and germination were negatively affected by exposure to acidic condition and bird internal temperature. Cysts of A. malmogiense were more sensitive than P. borgei to treatments and to the presence of digestive enzymes. Vegetative cells did not survive conditions of bird internal temperature and formed pellicle cysts when exposed to gizzard-like acid conditions. We show that dinoflagellate resting cysts serve as dispersal propagules through migratory birds. Assuming a retention time of viable cysts of 2-12 h to duck stomach conditions, cysts could be dispersed 150-800 km and beyond.

Contrasting prevalence of selection and drift in the community structuring of bacteria and microbial eukaryotes.

Whether or not communities of microbial eukaryotes are structured in the same way as bacteria is a general and poorly explored question in ecology. Here, we investigated this question in a set of planktonic lake microbiotas in Eastern Antarctica that represent a natural community ecology experiment. Most of the analysed lakes emerged from the sea during the last 6000 years, giving rise to waterbodies that originally contained marine microbiotas and that subsequently evolved into habitats ranging from freshwater to hypersaline. We show that habitat diversification has promoted selection driven by the salinity gradient in bacterial communities (explaining ∼ 72% of taxa turnover), while microeukaryotic counterparts were predominantly structured by ecological drift (∼72% of the turnover). Nevertheless, we also detected a number of microeukaryotes with specific responses to salinity, indicating that albeit minor, selection has had a role in the structuring of specific members of their communities. In sum, we conclude that microeukaryotes and bacteria inhabiting the same communities can be structured predominantly by different processes. This should be considered in future studies aiming to understand the mechanisms that shape microbial assemblages.

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

Applying landscape genetics to the microbial world.

Landscape genetics, which explicitly quantifies landscape effects on gene flow and adaptation, has largely focused on macroorganisms, with little attention given to microorganisms. This is despite overwhelming evidence that microorganisms exhibit spatial genetic structuring in relation to environmental variables. The increasing accessibility of genomic data has opened up the opportunity for landscape genetics to embrace the world of microorganisms, which may be thought of as 'the invisible regulators' of the macroecological world. Recent developments in bioinformatics and increased data accessibility have accelerated our ability to identify microbial taxa and characterize their genetic diversity. However, the influence of the landscape matrix and dynamic environmental factors on microorganism genetic dispersal and adaptation has been little explored. Also, because many microorganisms coinhabit or codisperse with macroorganisms, landscape genomic approaches may improve insights into how micro- and macroorganisms reciprocally interact to create spatial genetic structure. Conducting landscape genetic analyses on microorganisms requires that we accommodate shifts in spatial and temporal scales, presenting new conceptual and methodological challenges not yet explored in 'macro'-landscape genetics. We argue that there is much value to be gained for microbial ecologists from embracing landscape genetic approaches. We provide a case for integrating landscape genetic methods into microecological studies and discuss specific considerations associated with the novel challenges this brings. We anticipate that microorganism landscape genetic studies will provide new insights into both micro- and macroecological processes and expand our knowledge of species' distributions, adaptive mechanisms and species' interactions in changing environments.

Airborne Microalgae: Insights, Opportunities, and Challenges.

Airborne dispersal of microalgae has largely been a blind spot in environmental biological studies because of their low concentration in the atmosphere and the technical limitations in investigating microalgae from air samples. Recent studies show that airborne microalgae can survive air transportation and interact with the environment, possibly influencing their deposition rates. This minireview presents a summary of these studies and traces the possible route, step by step, from established ecosystems to new habitats through air transportation over a variety of geographic scales. Emission, transportation, deposition, and adaptation to atmospheric stress are discussed, as well as the consequences of their dispersal on health and the environment and state-of-the-art techniques to detect and model airborne microalga dispersal. More-detailed studies on the microalga atmospheric cycle, including, for instance, ice nucleation activity and transport simulations, are crucial for improving our understanding of microalga ecology, identifying microalga interactions with the environment, and preventing unwanted contamination events or invasions.

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype.

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north-eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra-strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large-scale biogeographic patterns in this species.

Temporal changes in population structure of a marine planktonic diatom.

A prevailing question in phytoplankton research addresses changes of genetic diversity in the face of huge population sizes and apparently unlimited dispersal capabilities. We investigated population genetic structure of the pennate planktonic marine diatom Pseudo-nitzschia multistriata at the LTER station MareChiara in the Gulf of Naples (Italy) over four consecutive years and explored possible changes over seasons and from year to year. A total of 525 strains were genotyped using seven microsatellite markers, for a genotypic diversity of 75.05%, comparable to that found in other Pseudo-nitzschia species. Evidence from Bayesian clustering analysis (BA) identified two genetically distinct clusters, here interpreted as populations, and several strains that could not be assigned with ≥ 90% probability to either population, here interpreted as putative hybrids. Principal Component Analysis (PCA) recovered these two clusters in distinct clouds with most of the putative hybrids located in-between. Relative proportions of the two populations and the putative hybrids remained similar within years, but changed radically between 2008 and 2009 and between 2010 and 2011, when the 2008-population apparently became the dominant one again. Strains from the two populations are inter-fertile, and so is their offspring. Inclusion of genotypes of parental strains and their offspring shows that the majority of the latter could not be assigned to any of the two parental populations. Therefore, field strains classified by BA as the putative hybrids could be biological hybrids. We hypothesize that P. multistriata population dynamics in the Gulf of Naples follows a meta-population-like model, including establishment of populations by cell inocula at the beginning of each growth season and remixing and dispersal governed by moving and mildly turbulent water masses.

Mendelian inheritance pattern and high mutation rates of microsatellite alleles in the diatom Pseudo-nitzschia multistriata.

The diatom Pseudo-nitzschia multistriata exhibits a diplontic life cycle composed of an extensive phase of vegetative cell division and a brief phase of sexual reproduction. To explore genotypic stability, we genotyped seven polymorphic microsatellite loci in 26 monoclonal strains over 3-16 months in a culture maintenance regime. Moreover, to assess inheritance patterns of the microsatellite alleles, we genotyped 246 F1 strains resulting from four mating experiments between parental strains of know genotype. Results generally conformed expectations according to Mendelian inheritance patterns, but deviations were detected indicating mutations during sexual reproduction. A total of forty-two mutations were detected in the clonal cultures over time. Microsatellites with more core-repeats accumulated mutations faster. The mutation rate varied significantly across loci and strains. A binomial mass function and a computer simulation showed that the mutation rate was significantly higher during the first months of culture (µ≈3×10(-3) per locus per cell division) and decreased to µ≈1×10(-3) in the strains kept for 16 months. Our results suggest that genetic mutations acquired in both the vegetative phase and sexual reproduction add to the allelic diversity of microsatellites, and hence to the genotypic variation present in a natural population.

Microsatellite primers in the planktonic diatom Pseudo-nitzschia multistriata (Bacillariophyceae).

PREMISE OF THE STUDY: Seven microsatellite loci were characterized for the toxic diatom Pseudo-nitzschia multistriata Takano (Takano) to investigate intraspecific variability and estimate population genetic structure over blooms, seasons, and sexual and vegetative reproduction. METHODS AND RESULTS: Selected microsatellites consisted of di- and trinucleotide repeats in the core region, and showed four to twelve alleles per locus in strains of P. multistriata collected in the Gulf of Naples (Italy). Primer pairs were species-specific since they positively amplified against conspecific strains from Portugal and Spain but failed to generate PCR products from the diatoms Pseudo-nitzschia pseudodelicatissima (Hasle) Hasle and Leptocylindrus minimum Gran. CONCLUSIONS: The seven selected microsatellite markers will be useful in studying population dynamics of Pseudo-nitzschia multistriata in space and time.