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Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships. more...

Microbially induced calcification is an ancient, community-driven mineralisation process that produces different types of microbialites. Symbiolites are photosynthesis-induced microbialites, formed by calcifying co-cultures of dinoflagellates from the family Symbiodiniaceae and bacteria. Symbiolites encase the calcifying community as endolithic cells, pointing at an autoendolithic niche of symbiotic dinoflagellates, and provide a rare opportunity to study the role of bacteria in bacterial-algal calcification, as symbiodiniacean cultures display either distinct symbiolite-producing (SP) or non-symbiolite-producing (NP) phenotypes. Using Illumina sequencing, we found that the bacterial communities of SP and NP cultures differed significantly in the relative abundance of 23 genera, 14 families, and 2 phyla. SP cultures were rich in biofilm digesters from the phylum Planctomycetes and their predicted metagenomes were enriched in orthologs related to biofilm formation. In contrast, NP cultures were dominated by biofilm digesters from the Bacteroidetes, and were inferred as enriched in proteases and nucleases. Functional assays confirmed the potential of co-cultures and bacterial isolates to produce biofilms and point at acidic polysaccharides as key stimulators for mineral precipitation. Hence, bacteria appear to influence symbiolite formation primarily through their biofilm-producing and modifying activity and we anticipate that symbiolite formation, as a low-complexity in vitro model, will significantly advance our understanding of photosynthesis-induced microbial calcification processes. more...

This protocol produces bacteria-depleted (“axenic”) Symbiodiniaceae cultures. The depletion level achieved using the following methods is comparable to what was previously described as an “axenic” culture (Bieri et al., 2016; Xiang et al., 2013). The present protocol was applied to 10 Symbiodiniaceae strains and proved successful in generating bacteria-depleted cultures after its application, for an extended period of time (up to 4 weeks), characterized by both culture-dependent and -independent techniques. Tested cultures showed normal growth and photosynthetic efficiency after recovery from the treatment. Variations of the protocol were developed to increase the success of bacterial depletion for different microalgal strains and to make it applicable to algal strains that do not grow well on solid media. Treatments can also be performed in 24-well plates in order to facilitate the measurement of Fv/Fmthrough the plate bottom using a fiber optic cable, in order to track photophysiology. This protocol was developed in parallel with a recently published method (Costa et al., 2019) to generate bacteria-depleted Aiptasia CC7 polyps, in order to have a toolbox at disposal to disassemble the different compartments of an anthozoan metaorganism for functional testing: the bacterial microbiome, the microalgal symbionts, and the anthozoan host animal. All steps of culture manipulation in this protocol must be done under a sterile environment, using common sterile work practices! more...

The Symbiodiniaceae are a family of marine dinoflagellates known mostly for their endosymbiotic interactions with invertebrates and protists, but facultatively and exclusively free-living life histories in this family are also evident. A recent systematic revision of the Symbiodiniaceae replaced the clade-based nomenclature of seven divergent lineages of "Symbiodinium" sensu lato with one based on formally described genera. The revised taxonomy was not extended to the whole group because type species to describe a new genus for each of the remaining clades and subclades were lacking. In an effort to characterize benthic habitats of symbiodiniaceans in sediments at Heron Island (Great Barrier Reef, Australia), we isolated >100 monoclonal Symbiodiniaceae cultures. Four of these belonged to Symbiodiniaceae 'subclade' Fr3, and three to Clade H, based on nucleotide sequence similarity (ITS2, LSU, cp23S, and mtCOB), representing the first cultures of these taxa. Based on these isolates, we propose two new genera: Freudenthalidium gen. nov. and Halluxium gen. nov., circumscribing Clades Fr3 and H, respectively. Three new species are described: Freudenthalidium heronense, F. endolithicum, and Halluxium pauxillum. Kofoidian tabulations of motile cells confirm previous observations that amphiesmal vesicle arrangements are generally conserved across the family. These descriptions are an important step toward completing the systematic revision of the Symbiodiniaceae. That this contribution was enabled by isolates from an endopsammic habitat highlights the potential of discovering new symbiodiniacean species in the environment, the study of which will lead to a deeper understanding of free-living versus symbiotic life histories in this ecologically important family of dinoflagellates. more...

A Gram-stain-negative, facultatively anaerobic, oxidase- and catalase-positive, rod-shaped bacterium, strain SYM1T, was isolated from a culture of Symbiodinium sp., an algal symbiont of the sea anemone Aiptasia tagetes collected in Puerto Rico. Growth was observed at 4–40 °C (optimum 30 °C), at pH 5.0–11.0 (optimum pH 8.0) and with 0.5–8 % (optimum 2 %) (w/v) NaCl. Phylogenetic analyses of 16S rRNA gene sequences showed that strain SYM1T was a member of the genus Neptunomonas with the type strain of Neptunomonas naphthovorans as the closest phylogenetic relative with a pairwise sequence similarity of 98.15 %. However, DNA–DNA relatedness between strain SYM1T and N. naphthovorans CIP 106451T was 24 %. Moreover, strain SYM1T could be distinguished from its closest relative by several phenotypic characteristics such as NaCl, pH and temperature tolerance, nitrate reduction and utilization of carbon substrates. The major cellular fatty acids were C16 : 0, C18 : 1ω7c and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 0 2-OH). The genomic DNA G+C content of strain SYM1T was 45 mol%. Ubiquinone-8 (Q-8) was the only respiratory quinone detected. Based on a polyphasic taxonomic characterization, strain SYM1T represents a novel species of the genus Neptunomonas , for which the name Neptunomonas phycophila sp. nov. is proposed. The type strain is SYM1T ( = LMG 28329T = CECT 8716T). more...

Dinoflagellates of the genus Symbiodinium live in symbiosis with many invertebrates, including reef-building corals. Hosts maintain this symbiosis through continuous regulation of Symbiodinium cell density via expulsion and degradation (postmitotic) and/or constraining cell growth and division through manipulation of the symbiont cell cycle (premitotic). Importance of premitotic regulation is unknown since little data exists on cell cycles for the immense genetic diversity of Symbiodinium. We therefore examined cell cycle progression for several distinct SymbiodiniumITS2-types (B1, C1, D1a). All types exhibited typical microalgal cell cycle progression, G1 phase through to S phase during the light period, and S phase to G2 /M phase during the dark period. However, the proportion of cells in these phases differed between strains and reflected differences in growth rates. Undivided larger cells with 3n DNA content were observed especially in type D1a, which exhibited a distinct cell cycle pattern. We further compared cell cycle patterns under different growth light intensities and thermal regimes. Whilst light intensity did not affect cell cycle patterns, heat stress inhibited cell cycle progression and arrested all strains in G1 phase. We discuss the importance of understanding Symbiodinium functional diversity and how our findings apply to clarify stability of host-Symbiodinium symbioses. more...

In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high-resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two-step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1-5.8S-ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species. more...

Significance The dinoflagellate genus Symbiodinium is best known for harboring important endosymbiotic algae of marine invertebrates, notably reef-building corals. However, these dinoflagellates also live freely within coral reef waters and sediments and provide an important environmental pool for the colonization of new coral recruits. Although Symbiodinium facilitate coral calcification indirectly when in hospite , we show that they also can calcify in partnership with bacteria when free living. This discovery offers entirely new perspectives on fundamental questions regarding the life cycle and ecology of these dinoflagellates and could help explain how changes in ocean chemistry created a selective pressure that ultimately led Symbiodinium to establish an endosymbiotic life style. To our knowledge, our findings document the first identified dinoflagellate–bacterial calcifying community. more...

Summary: Numerous software applications exist to deal with synthetic gene design, granting the field of heterologous expression a significant support. However, their dispersion requires the access to different tools and online services in order to complete one single project. Analyzing codon usage, calculating codon adaptation index (CAI), aligning orthologs and optimizing genes are just a few examples. A software application, EuGene, was developed for the optimization of multiple gene synthetic design algorithms. In a seamless automatic form, EuGene calculates or retrieves genome data on codon usage (relative synonymous codon usage and CAI), codon context (CPS and codon pair bias), GC content, hidden stop codons, repetitions, deleterious sites, protein primary, secondary and tertiary structures, gene orthologs, species housekeeping genes, performs alignments and identifies genes and genomes. The main function of EuGene is analyzing and redesigning gene sequences using multi-objective optimization techniques that maximize the coding features of the resulting sequence. Availability: EuGene is freely available for non-commercial use, at http://bioinformatics.ua.pt/eugene Contact: paulogaspar@ua.pt more...

Background: Codon pair usage (codon context) is a species specific gene primary structure feature whose evolutionary and functional roles are poorly understood. The data available show that codon-context has direct impact on both translation accuracy and efficiency, but one does not yet understand how it affects these two translation variables or whether context biases shape gene evolution. Methodologies/Principal findings: Here we study codon-context biases using a set of 72 orthologous highly conserved genes from bacteria, archaea, fungi and high eukaryotes to identify 7 distinct groups of codon context rules. We show that synonymous mutations, i.e., neutral mutations that occur in synonymous codons of codon-pairs, are selected to maintain context biases and that non-synonymous mutations, i.e., non-neutral mutations that alter protein amino acid sequences, are also under selective pressure to preserve codon-context biases. Conclusions: Since in vivo studies provide evidence for a role of codon context on decoding fidelity in E. coli and for decoding efficiency in mammalian cells, our data support the hypothesis that, like codon usage, codon context modulates the evolution of gene primary structure and fine tunes the structure of open reading frames for high genome translational fidelity and efficiency in the 3 domains of life. published more...