A case for an active eukaryotic marine biosphere during the Proterozoic era.

The microfossil record demonstrates the presence of eukaryotic organisms in the marine ecosystem by about 1,700 million years ago (Ma). Despite this, steranes, a biomarker indicator of eukaryotic organisms, do not appear in the rock record until about 780 Ma in what is known as the "rise of algae." Before this, it is argued that eukaryotes were minor ecosystem members, with prokaryotes dominating both primary production and ecosystem dynamics. In this view, the rise of algae was possibly sparked by increased nutrient availability supplying the higher nutrient requirements of eukaryotic algae. Here, we challenge this view. We use a size-based ecosystem model to show that the size distribution of preserved eukaryotic microfossils from 1,700 Ma and onward required an active eukaryote ecosystem complete with phototrophy, osmotrophy, phagotrophy, and mixotrophy. Model results suggest that eukaryotes accounted for one-half or more of the living biomass, with eukaryotic algae contributing to about one-half of total marine primary production. These ecosystems lived with deep-water phosphate levels of at least 10% of modern levels. The general lack of steranes in the pre-780-Ma rock record could be a result of poor preservation.

Interspecific Competition Study Between Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae)-Two Ichthyotoxic Species that Co-occur in Scandinavian Waters.

The genus Pseudochattonella has become a frequent component of late winter-early spring phytoplankton community in Scandinavian waters, causing extensive fish kills and substantial economic losses. One of currently two recognised species, P. farcimen, is often abundant prior to the diatom spring bloom. Recent field studies have revealed that P. farcimen and P. verruculosa have a period of overlap in their temperature ranges and thus their seasonal occurrences. Using laboratory cultures, we investigated the seasonal succession and growth of P. farcimen and P. verruculosa in both mono- and mixed-culture using the recently developed Pseudochattonella 'qPCR subtraction method', which for the first time allowed the simultaneous enumeration of these morphologically indistinguishable species in mixed assemblages. We examined how these species interacted over four different temperatures (5, 8, 11 and 15 °C). The observed growth rates and cell yields varied with temperature revealing their preferred temperature optima. P. farcimen was able to achieve positive net growth over all temperatures, while P. verruculosa failed to grow below 11 °C. Growth responses were statistically different between mono- and mixed-cultures with the outcome of these interactions being temperature-dependent. Nutrients (nitrate and phosphate) and pH levels were also measured throughout the growth experiments to better understand how these factors influenced growth of both species. P. verruculosa was shown to be less sensitive to high pH as growth ceased at pH 9.1, whereas P. farcimen stopped growing at pH 8.4. Understanding the influence of abiotic factors (e.g. temperature, pH and competition) on growth rates allows for a better understanding and prediction of phytoplankton community dynamics.

A quantitative real-time PCR assay for identification and enumeration of the occasionally co-occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures.

The ichthyotoxic genus Pseudochattonella forms recurrent extensive blooms in coastal waters in Japan, New Zealand and Northern Europe. It comprises of two morphologically similar species, P. verruculosa and P. farcimen, which complicates visual species identification and enumeration of live and fixed material. Primers designed previously could not quantitatively distinguish species in mixed assemblages. To address this issue we developed two primer sets: one revealed itself to be genus specific for Pseudochattonella and the other species-specific for P. verruculosa. By subtracting cell estimates for P. verruculosa from combined results we could calculate cell numbers for P. farcimen. This approach has overcome the challenges posed by the very limited sequence availability and low gene variability between the two species. The qPCR assay was extensively tested for specificity, efficiency and sensitivity over an entire growth cycle in both single and mixed assemblages. Comparison of cell abundance estimates obtained by qPCR assay and microscopy showed no statistically significant difference until stationary and death phases. The assay was also tested on environmental samples collected during a small Pseudochattonella bloom in Denmark in March-April 2015. It was impossible to distinguish P. farcimen and P. verruculosa by light microscopy but qPCR showed both species were present. The two methods provided nearly identical cell numbers but the assay provided discrimination and enumeration of both species.

The competitive dynamics of toxic Alexandrium fundyense and non-toxic Alexandrium tamarense: The role of temperature.

The dinoflagellate Alexandrium produces paralytic shellfish poisoning toxins. The genus is globally distributed, with Scottish waters being of particular interest due to the co-occurrence of different species and strains. In Scottish waters, Alexandrium was historically thought to be dominated by the highly toxic (Group I) Alexandrium fundyense. However, the morphologically indistinguishable (Group III) Alexandrium tamarense has recently also been found to co-occur, raising important questions in relation to Alexandrium biogeography. To begin to address these, we investigated Alexandrium growth, yield and toxin production in a range of temperature conditions characteristics of present and potential future conditions, using a recently developed flow cytometry method that allowed, for the first time, simultaneous enumeration of the cryptic species in co-culture. Experiments were undertaken in a range of temperatures (12, 15, 18 and 21°C) in the phosphate (P) limiting conditions that promotes A. fundyense toxicity. Cell/biomass yield was greater for A. tamarense at all temperatures, with observed growth rates varying with temperature. Growth rather and yield were different in mono- and co-culture with the outcome of these interactions also being temperature dependent. For toxic A. fundyense, GTX-3, STX and NEO were the dominant analogues, but total toxicity, toxicity per cell and the number of, and relative proportion of, toxin analogues changed in relation to the onset of P limitation and also as a function of temperature, with the highest toxin concentrations per cell being observed at 12°C. Toxin concentrations were approximately double in P limited stationary phase compared to exponential growth. Toxin concentrations were lower in the co-cultures, indicating inhibition of production in the presence of non-toxic A. tamarense. The strong performance of A. tamarense is in co-culture at odds with the historical understanding that Scottish waters were dominated by A. fundyense and indicates that changes in water temperatures, and also potentially alleopathic interactions, will influence Alexandrium populations and hence the PSP toxicity threat to humans from shellfish.

Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species.

Harmful algal blooms (HAB) pose serious economic and health risks worldwide. Current methods of identification require high levels of taxonomic skill and can be highly time-consuming thus limiting sample throughput. So, new rapid and reliable methods for detection and enumeration of HAB species are required. Here we describe a high-throughput, multiplex-qPCR (M-qPCR) method using hydrolysis probe technology for the simultaneous detection of four HAB species commonly found in many coastal areas worldwide: Alexandrium tamarense, Karenia mikimotoi, Karlodinium veneficum and Prymnesium parvum. Primers and probes were species-specific and highly efficient when tested in simplex. Species were then added in succession and the assay conditions adjusted until all four species could be quantitatively evaluated simultaneously. Enumeration accuracy of the M-qPCR assay as a monitoring tool was evaluated using spiked natural environmental samples from Danish coastal waters. Comparison of estimates of cell abundances obtained by the M-qPCR technique with those obtained by light microscopy (Sedgwick Rafter technique) showed no statistically significant difference across a range of concentrations. We were also able to identify and enumerate target cells that would be below the detection limit of light microscopy making this a suitable method for early bloom detection or for low biomass species. With the development of molecular probes for a greater number of algal species M-qPCR will be of great benefit to phytoplankton monitoring programmes and the aquaculture industry worldwide.