Predictability of thermal fluctuations influences functional traits of a cosmopolitan marine diatom

Evolutionary theory predicts that organismal plasticity should evolve in environments that fluctuate regularly. However, in environments that fluctuate less predictably, plasticity may be constrained because environmental cues become less reliable for expressing the optimum phenotype. Here, we examine how the predictability of +5°C temperature fluctuations impacts the phenotype of the marine diatom Thalassiosira pseudonana. Thermal regimes were informed by temperatures experienced by microbes in an ocean simulation and featured regular or irregular temporal sequences of fluctuations that induced mild physiological stress. Physiological traits (growth, cell size, complexity and pigmentation) were quantified at the individual cell level using flow cytometry. Changes in cellular complexity emerged as the first impact of predictability after only 8–11 days, followed by deleterious impacts on growth on days 13–16. Specifically, cells with a history of irregular fluctuation exposure exhibited a 50% reduction in growth compared with the stable reference environment, while growth was 3–18 times higher when fluctuations were regular. We observed no evidence of heat hardening (increasingly positive growth) with recurrent fluctuations. This study demonstrates that unpredictable temperature fluctuations impact this cosmopolitan diatom under ecologically relevant time frames, suggesting shifts in environmental stochasticity under a changing climate could have widespread consequences among ocean primary producers

A high-throughput assay for quantifying phenotypic traits of microalgae

High-throughput methods for phenotyping microalgae are in demand across a variety of research and commercial purposes. Many microalgae can be readily cultivated in multi-well plates for experimental studies which can reduce overall costs, while measuring traits from low volume samples can reduce handling. Here we develop a high-throughput quantitative phenotypic assay (QPA) that can be used to phenotype microalgae grown in multi-well plates. The QPA integrates 10 low-volume, relatively high-throughput trait measurements (growth rate, cell size, granularity, chlorophyll a, neutral lipid content, silicification, reactive oxygen species accumulation, and photophysiology parameters: ETRmax, Ik, and alpha) into one workflow. We demonstrate the utility of the QPA on Thalassiosira spp., a cosmopolitan marine diatom, phenotyping six strains in a standard nutrient rich environment (f/2 media) using the full 10-trait assay. The multivariate phenotypes of strains can be simplified into two dimensions using principal component analysis, generating a trait-scape. We determine that traits show a consistent pattern when grown in small volume compared to more typical large volumes. The QPA can thus be used for quantifying traits across different growth environments without requiring exhaustive large-scale culturing experiments, which facilitates experiments on trait plasticity. We confirm that this assay can be used to phenotype newly isolated diatom strains within 4 weeks of isolation. The QPA described here is highly amenable to customisation for other traits or unicellular taxa and provides a framework for designing high-throughput experiments. This method will have applications in experimental evolution, modelling, and for commercial applications where screening of phytoplankton traits is of high importance

Multitrait diversification in marine diatoms in constant and warmed environments

Phytoplankton are photosynthetic marine microbes that affect food webs, nutrient cycles and climate regulation. Their roles are determined by correlated phytoplankton functional traits including cell size, chlorophyll content and cellular composition. Here, we explore patterns of evolution in interrelated trait values and correlations. Because both chance events and natural selection contribute to phytoplankton trait evolution, we used population bottlenecks to diversify six genotypes of Thalassiosirid diatoms. We then evolved them as large populations in two environments. Interspecific variation and within-species evolution were visualized for nine traits and their correlations using reduced axes (a trait-scape). Our main findings are that shifts in trait values resulted in movement of evolving populations within the trait-scape in both environments, but were more frequent when large populations evolved in a novel environment. Which trait relationships evolved was population-specific, but greater departures from ancestral trait correlations were associated with lower population growth rates. There was no single master trait that could be used to understand multi-trait evolution. Instead, repeatable multi-trait evolution occurred along a major axis of variation defined by several diatom traits and trait relationships. Because trait-scapes capture changes in trait relation-ships and values together, they offer an insightful way to study multi-trait variatio

The evolution of trait correlations constrains phenotypic adaptation to high CO 2 in a eukaryotic alga

Microbes form the base of food webs and drive biogeochemical cycling. Predicting the effects of microbial evolution on global elemental cycles remains a significant challenge due to the sheer number of interacting environmental and trait combinations. Here, we present an approach for integrating multivariate trait data into a predictive model of trait evolution. We investigated the outcome of thousands of possible adaptive walks parameterized using empirical evolution data from the alga Chlamydomonas exposed to high CO(2). We found that the direction of historical bias (existing trait correlations) influenced both the rate of adaptation and the evolved phenotypes (trait combinations). Critically, we use fitness landscapes derived directly from empirical trait values to capture known evolutionary phenomena. This work demonstrates that ecological models need to represent both changes in traits and changes in the correlation between traits in order to accurately capture phytoplankton evolution and predict future shifts in elemental cycling

The ecology and toxin production of gambierdiscus and fukuyoa species from the Pacific

Ciguatera fish poisoning is a common food-borne illness experienced in tropical locations worldwide. It is caused by the ingestion of fish and seafood that have accumulated ciguatoxins (CTXs) (and possibly maitotoxins, MTXs) produced by microalgal dinoflagellates from the genera Gambierdiscus and Fukuyoa. This thesis firstly investigated the growth and ciguatoxin production by a Gambierdiscus polynesiensis strain from the Cook Islands using laboratory based studies. The effect of growth phase, nutrient regime, trace metals, and growth vessel were examined. Secondly, ecological studies were conducted around Tonga, a Pacific island nation affected by ciguatera, to establish diversity and abundance of Gambierdiscus and Fukuyoa spp. using molecular and microscopy tools. Relationships between microalgae assemblages and their surrounding habitats were also evaluated. The culturing experiments contained in Chapter 2 showed that G. polynesiensis cultures reached stationary phase faster under a lower nutrient regime of f2 media:seawater 1:3 ratio when compared with 1:2 ratio. Cumulatively, all 4 monitored CTX congeners (CTX-3B, -3C, -4A and -4B) mirrored the growth curve, increasing from lag through exponential phase and peaking at stationary phase. CTX-3B was present in the highest concentrations across all time points. When nitrate concentrations in the growth media were manipulated, low N:P ratios yielded lower cells densities but higher CTXs per cell. An inverse relationship between densities and CTXs per cell was observed with the reverse result observed at high N:P ratios. When phosphate concentrations were manipulated, high N:P ratios led to higher CTXs per cell and lower cell densities with the an inverse relationship observed between the two variables. Both flat tissue culture flasks and plastic culture bags were effective for growth of G. polynesiensis but did not yield higher cell densities regardless of their higher surface area. This study shows the potential effects of the environment on CTX production in G. polynesiensis as well as the strain-specific toxin production of this Cook Islands strain. These data may also inform protocols for bulk culture of Gambierdiscus for the purposes of toxin purification. The third chapter of this thesis examined the diversity of Gambierdiscus and Fukuyoa species in seagrass and algae dominated habitats around the island of Tongatapu, Tonga. Water quality measures and habitat surveys were conducted at four sites around the island and microalgae collected on artificial substrates and off macrophytes. Macrophytes from all four sites supported Gambierdiscus growth. Genus and species-level real-time PCR assays were used to identify the presence of Gambierdiscus and Fukuyoa species across the sites. All artificial substrates and macrophyte samples tested positive for the presence of the genus, with the exception of the artificial substrates at Ha’ateiho. In addition, G. carpenteri, G. pacificus and G. honu were detected in both algal and seagrass habitats at Sopu, Fafa, and Kolonga, with G. australes observed only in the algae habitat at all three sites. In contrast, F. paulensis was observed only in seagrass beds at Sopu; F. ruetzleri was observed only in seagrass and Sopu and algae beds at Fafa. Overall, the species-specific detection from macrophyte samples was low when compared to the artificial substrates. Live cells isolated were identified as G. australes, G. pacificus and G. honu as well as one Ostreopsis species. All isolates produced toxins. There was high diversity of Gambierdiscus species around Tongatapu and both seagrass and algae beds were suitable for Gambierdiscus growth. The efficacy of artificial substrates as a sampling tool for benthic dinoflagellates was evaluated and advantages and disadvantages of using real-time PCR for ecological studies are discussed. The fourth study examined microalgae diversity and abundance in Ha’apai and Vava’u island groups of Tonga as well as repeated surveys of Tongatapu. Nineteen sites were chosen across the three groups including two off-shore islands in Tongatapu. Shallow sub-tidal habitats were classified either as seagrass, algae, Halimeda, or turf dominant. Habitats were surveyed using quadrats and water quality measures taken in each habitat. Two lagoon sites were also sampled for microalgae but not surveyed with quadrats due to high turbidity. Microalgae were collected via settlement on artificial substrates or by collecting macrophytes. One additional substrate at each point was deployed to collect live samples for cell isolations. Gambierdiscus, Ostreopsis, and Prorocentrum cells were identified from each sample and counted using light microscopy. Prorocentrum was the most abundant genus in almost every sample. Gambierdiscus was more abundant than Ostreopsis but there was often high variability within habitats. A metabarcoding approach was used to evaluate Gambierdiscus and Fukuyoa species diversity. DNA was extracted from all samples and amplified using PCR with two primer pairs, one targeting the V4 region of the 18S SSU gene and the other the D1-D2 region of the 28S LSU gene. Cleaned PCR products were sent for sequencing using Illumina MiSeqTM sequencing and returned sequences analysed using bioinformatic processing. The highest Gambierdiscus and Fukuyoa species diversity was found in Ha’apai where eight species were detected using this technique. There was high variation in Gambierdiscus diversity between and within sites and habitats, with the exception of Vava’u where Gambierdiscus was detected in only four samples. The dominant species in Vava’u was G. pacificus, with 2 other species found only in one sample. The dominant species in Tongatapu was G. carpenteri, with G. pacificus and G. cheloniae also observed at a number of sites. A recently identified new species currently classified ‘UTSHI6A6’ in GenBank was found in both Ha’apai and Tongatapu. The LSU gene amplification method was more sensitive than the SSU amplification in detecting Gambierdiscus and Fukuyoa species and had higher resolution due to the higher availability of sequences for the LSU gene region. This study shows the potential of metabarcoding as a tool for assessing Gambierdiscus and Fukuyoa species diversity from coastal tropical environments and shows that these genera are common and diverse in a variety of habitats around Tonga. The final chapter of this thesis summarises the overall goals of ecologists in the field of ciguatera research. The applications and limitations of culturing and field-based studies are evaluated as well as the future research needs. The factors that should be considered when developing a microalgae based monitoring programme are discussed. This thesis established new knowledge regarding toxin production of Gambierdiscus polynesiensis in culture and is the first direct record of ciguatera-related dinoflagellates in Tonga