Comparison of Spatial and Temporal Genetic Differentiation in a Harmful Dinoflagellate Species Emphasizes Impact of Local Processes

Population genetic studies provide insights into intraspecific diversity and dispersal patterns of microorganisms such as protists, which help understanding invasions, harmful algal bloom development and occurrence of seafood poisoning. Spatial genetic differentiation has been reported in many microbial species indicating significant dispersal barriers among different habitats. Temporal differentiation has been less studied and its frequency, drivers, and magnitude are thus relatively poorly understood. The toxic dinoflagellate species Gambierdiscus caribaeus was sampled during 2 years in the Florida Keys, and repeatedly from 2006 to 2016 at St. Thomas, US Virgin Islands (USVI), including a 3-year period with monthly sampling, enabling a comparison of spatial and temporal genetic differentiation. Samples from the USVI site showed high temporal variability in local population structure, which correlated with changes in salinity and benthic habitat cover. In some cases, temporal variability exceeded spatial differentiation, despite apparent lack of connectivity and dispersal across the Greater Caribbean Region based on the spatial genetic data. Thus, local processes such as selection might have a stronger influence on population structure in microorganisms than geographic distance. The observed high temporal genetic diversity challenges the prediction of harmful algal blooms and toxin concentrations, but illustrates also the evolutionary potential of microalgae to respond to environmental change

AFLP data based on presence (1) and absence (0) of AFLP loci (A4-F171) in Gonyostomum semen

Many recent studies have found genetically differentiated populations in microorganisms despite potentially high dispersal. We designed a study to specifically examine the importance of physical dispersal barriers, i.e. geographic distance and lack of hydrological connectivity, in restricting gene flow and enhancing divergence in limnic microorganisms. We focused on the nuisance microalga Gonyostomum semen, which has recently expanded in northern Europe and differentiated into genetically distinct populations. Gonyostomum semen was sampled from six lakes distributed in two adjacent watersheds, which thereby comprised, both connected and non-connected lakes. The individual isolates were genotyped by Amplified Fragment Length Polymorphism. Several lake populations were differentiated from each other, but connectivity within watersheds could not explain the observed population genetic pattern. However, isolation by distance was moderate and might limit the gene flow among distant populations. In addition, we found low, but significant linkage disequilibrium, which indicates regular sexual recombination in this species, despite its high degree of asexual reproduction. Therefore, we conclude that the genetic properties of microalgae with occasional sexual reproduction essentially mirror regularly recombining species. Furthermore, the data indicated bottlenecks supporting the hypothesized recent range expansion of this species

Sequencing and phylogenetic analysis of chloroplast genes in freshwater raphidophytes

The complex evolution of chloroplasts in microalgae has resulted in highly diverse pigment profiles. Freshwater raphidophytes, for example, display a very different pigment composition to marine raphidophytes. To investigate potential differences in the evolutionary origin of chloroplasts in these two groups of raphidophytes, the plastid genomes of the freshwater species Gonyostomum semen and Vacuolaria virescens were sequenced. To exclusively sequence the organelle genomes, chloroplasts were manually isolated and amplified using single-cell whole-genome-amplification. Assembled and annotated chloroplast genes of the two species were phylogenetically compared to the marine raphidophyte Heterosigma akashiwo and other evolutionarily more diverse microalgae. These phylogenetic comparisons confirmed the high relatedness of all investigated raphidophyte species despite their large differences in pigment composition. Notable differences regarding the presence of light-independent protochlorophyllide oxidoreductase (LIPOR) genes among raphidophyte algae were also revealed in this study. The whole-genome amplification approach proved to be useful for isolation of chloroplast DNA from nuclear DNA. Although only approximately 50% of the genomes were covered, this was sufficient for a multiple gene phylogeny representing large parts of the chloroplast genes

Microsatellite markers for the dinoflagellate Gambierdiscus caribaeus from high-throughput sequencing data

Members of the benthic dinoflagellate genus Gambierdiscus are the causative agents of ciguatera fish poisoning worldwide. Ciguatera outbreaks appear to be more common in recent years and new incidences are reported from unprecedented regions. To investigate Gambierdicus population dynamics, connectivity, and dispersal routes, we developed microsatellite markers for Gambierdiscus caribaeus, a globally distributed species that is common at our study site at St. Thomas in the US Virgin Islands. We used high-throughput partial genome sequencing along with an existing transcriptome for microsatellite discovery. Screening of contigs with less than three times coverage resulted in 558 (partial genome) and 33 (transcriptome) candidate microsatellites. Four primer pairs from the partial genome and three from the transcriptome successfully amplified polymorphic microsatellites in multiplexed PCR reactions. The seven markers were tested on 150 G. caribaeus strains isolated monthly from August 2013 to July 2015 at St. Thomas, USVI. The numbers of alleles per locus varied between 3 and 14, and the allele diversity ranged from 0.214 to 0.899 in this dataset. These newly developed microsatellites will enable studies of population structure, connectivity, and dispersal in G. caribaeus and can give new insights into the expansion of ciguatera outbreaks worldwide

Infection strategies of different chytrids in a diatom spring bloom

Diatom blooms are often accompanied by an increase in parasitic chytrids that kill the host cells, which they are infecting, and can contribute to the decline of the bloom. However, host specificity and range of these chytrids are currently poorly understood. Low host specificity would enable the parasites to opportunistically infect any diatom species, while specialisation on infecting specific high-biomass species could result in high prevalence and rapid spread of infection. We investigated such host-parasite interactions by monitoring the diverse diatom spring bloom in lake Erken using amplicon sequencing. We also performed infection experiments with two different, newly isolated chytrid species and several diatom cultures from the bloom. Chytridiomycota displayed the highest relative abundance of all parasitic lineages and were probably physically attached to larger organisms. Since the chytrids reached maximum abundance shortly after a peak in diatom reads, they were probably infecting these important primary producers. Phylogenetic analyses of the isolated chytrid strains identified them as members of the classes Rhizophydiales and Lobulomycetales. The infection experiments revealed high host specificity in these two chytrids targeting different diatom species. The experimental results supported statistical analyses of the environmental sequencing data, which suggested the presence of two different infection strategies: the most abundant chytrid species were specialised on infecting dominant diatom genera (i.e. Stephanodiscus, Aulacoseira, Asterionella), while rarer chytid species infected a range of less abundant diatoms

Count data of germinated Gonyostomum semen cells and subsequent cell divisions

Colonization of new habitats through dispersal of phytoplankton cysts might be limited, if resident populations outcompete invaders during germination. We reciprocally transferred Gonyostomum semen (Raphidophyceae) cysts from three lakes into native and foreign waters originating from the respective habitats. Germination rate and germling growth were impacted by water origin, but there was no preference for native water. Gonyostomum semen's ability to germinate in different conditions might explain its expansion in northern Europe