Plankton biodiversity and species co-occurrence based on environmental DNA – a multiple marker study

Metabarcoding in combination with high-throughput sequencing (HTS) allows simultaneous detection of multiple taxa by targeting single or several taxonomically informative gene regions from environmental DNA samples. In this study, a multiple-marker HTS approach was applied to investigate the plankton diversity and seasonal succession in the Baltic Sea from winter to autumn. Four different markers targeting the 16S, 18S, and 28S ribosomal RNA genes were employed, including a marker for more efficient dinoflagellate detection. Typical seasonal changes were observed in phyto- and bacterioplankton communities. In phytoplankton, the appearance patterns of selected common, dominant, or harmful species followed the patterns also confirmed based on 20 years of phytoplankton monitoring data. In the case of zooplankton, both macro- and microzooplankton species were detected. However, no seasonal patterns were detected in their appearance. In total, 15 and 2 new zoo- and phytoplankton species were detected from the Baltic Sea. HTS approach was especially useful for detecting microzooplankton species as well as for investigating the co-occurrence and potential interactions of different taxa. The results of this study further exemplify the efficiency of metabarcoding for biodiversity monitoring and the advantage of employing multiple markers through the detection of species not identifiable based on a single marker survey and/or by traditional morphology-based methods

Reconstructing salinity changes and environmental influence on dinoflagellate cysts in the central Baltic Sea since the late 19th century

We present a record of dinoflagellate cyst assemblage composition, abundance, and morphology from the central Baltic Sea, spanning from the late 19th to the early 21st century. Environmental time-series were analyzed in relation to changes in community structure and diversity as inferred from the sediment record, and average summer sea surface salinity (SSS) was reconstructed based on the average process length of Protoceratium reticulatum resting cysts. The reconstructed summer SSS was compared to instrumental data for a critical evaluation of this approach. The most abundant species in this record were P. reticulatum and Biecheleria baltica, and on average ten taxa were identified per sample. The cyst record of B. baltica indicated that although this species has been present in the Gotland Basin at least since the 1880s, its concentrations have increased significantly since the 1980s, possibly linked to eutrophication. Variations in assemblage composition and P. reticulatum cyst morphology reflected patterns of major, instrumentally recorded hydrographic and environmental changes in the Baltic Sea during the past century. The variability in microfossil relative abundances was best explained by the average spring SSS as well as by the average NO3 concentrations during spring and by the combined effects of average summer SSS and NAO index variability. Reconstructed summer SSS and instrumental SSS showed notable differences, depending on the year and function applied for reconstruction. Although roughly reflecting the same patterns, the reconstructed values are offset when compared to instrumental measurements. We put forward suggestions for improvement of the process-length method and recommend using the reconstructed values as an indication of relative changes in past summer sea surface salinity, preferably as part of a multiproxy approach

Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding

Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p < 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions

Monitoring harmful microalgal species and their appearance in Tokyo Bay, Japan, using metabarcoding

During the recent decade, high-throughput sequencing (HTS) techniques, in particular, DNA metabarcoding, have facilitated increased detection of biodiversity, including harmful algal bloom (HAB) species. In this study, the presence of HAB species and their appearance patterns were investigated by employing molecular and light microscopy-based monitoring in Tokyo Bay, Japan. The potential co-appearance patterns between the HAB species, as well as with other eukaryotes and prokaryotes were investigated using correlation and association rule-based time-series analysis. In total, 40 unique HAB species were detected, including 12 toxin-producing HAB species previously not reported from the area. More than half of the HAB species were present throughout the sampling season (summer to autumn) and no structuring or succession patterns associated with the environmental conditions could be detected. Statistically significant (p < 0.05, rS ranging from −0.88 to 0.90) associations were found amongst the HAB species and other eukaryotic and prokaryotic species, including genera containing growth-limiting bacteria. However, significant correlations between species differed amongst the years, indicating that variability in environmental conditions between the years may have a stronger influence on the microalgal community structure and interspecies interactions than the variability during the sampling season. The association rule-based time-series analysis allowed the detection of a previously reported negative relationship between Synechococcus sp. and Skeletonema sp. in nature. Overall, the results support the applicability of metabarcoding and HTS-based microalgae monitoring, as it facilitates more precise species identification compared to light microscopy, as well as provides input for investigating potential interactions amongst different species/groups through simultaneous detection of multiple species/genera

DNA metabarcoding : Guidelines to monitor phytoplankton diversity and distribution in marine and brackish waters

We present guidelines for using environmental DNA metabarcoding, together with conventional techniques, to monitor the taxonomic diversity of phytoplankton in marine and brackish waters. The focus is on eukaryotic and prokaryotic phytoplankton using 18S and 16S rRNA primers and high-throughput sequencing. Information and recommendations on sampling, sample processing, molecular biological work, quality control, bioinformatics, data storage and management and cost estimates are included so that the method can be used to complement standardized light microscopy. A scientific literature review, discussion on future perspectives, reference databases and standardization are included. Using eDNA metabarcoding to complement standardized light microscopy advances conventional monitoring and research on phytoplankton communities to assess biodiversity and the state of the marine environment

Physical barriers and environmental gradients cause spatial and temporal genetic differentiation of an extensive algal bloom

Aim: To test if a phytoplankton bloom is panmictic, or whether geographical and environmental factors cause spatial and temporal genetic structure. Location: Baltic Sea. Method: During four cruises, we isolated clonal strains of the diatom Skeletonema marinoi from 9 to 10 stations along a 1132 km transect and analysed the genetic structure using eight microsatellites. Using F-statistics and Bayesian clustering analysis we determined if samples were significantly differentiated. A seascape approach was applied to examine correlations between gene flow and oceanographic connectivity, and combined partial Mantel test and RDA based variation partitioning to investigate associations with environmental gradients. Results: The bloom was initiated during the second half of March in the southern and the northern- parts of the transect, and later propagated offshore. By mid-April the bloom declined in the south, whereas high phytoplankton biomass was recorded northward. We found two significantly differentiated populations along the transect. Genotypes were significantly isolated by distance and by the south-north salinity gradient, which illustrated that the effects of distance and environment were confounded. The gene flow among the sampled stations was significantly correlated with oceanographic connectivity. The depletion of silica during the progression of the bloom was related to a temporal population genetic shift. Main conclusions: A phytoplankton bloom may propagate as a continuous cascade and yet be genetically structured over both spatial and temporal scales. The Baltic Sea spring bloom displayed strong spatial structure driven by oceanographic connectivity and geographical distance, which was enhanced by the pronounced salinity gradient. Temporal transition of conditions important for growth may induce genetic shifts and different phenotypic strategies, which serve to maintain the bloom over longer periods