High-throughput sequencing of the 16S rRNA gene on the Illumina platform is commonly used to assess microbial diversity in environmental samples. The MiniSeq, Illumina's latest benchtop sequencer, enables more cost-efficient DNA sequencing relative to larger Illumina sequencing platforms (e.g., MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high-throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed additional sequencing primers that enabled application of a dual-index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples in four different sequencing runs as a quality control benchmark. We were able to recapture a realistic richness of the mock community in all sequencing runs, and identify meaningful differences in alpha and beta diversity in the environmental samples. Furthermore, rarefaction analysis indicated diversity in many environmental samples was close to saturation. These results show that the MiniSeq can produce similar quantities of high-quality V4 reads compared to the MiSeq, yet is a cost-effective option for any laboratory interested in performing high-throughput 16S rRNA gene sequencing.

27High-throughput sequencing of the 16S rRNA gene is widely used in microbial ecology, with 45show that the MiniSeq can produce similar quantities of high quality V4 reads compared to the 46MiSeq. In addition, our protocol increases feasibility for small laboratories to perform their own 47 high-throughput sequencing of the 16S rRNA marker gene.

A general trend observed in animal skeletomes—the proteins occluded in animal skeletons—is the copresence of taxonomically widespread and lineage-specific proteins that actively regulate the biomineralization process. Among cnidarians, the skeletomes of scleractinian corals have been shown to follow this trend. However, distributions and phylogenetic analyses of biomineralization-related genes are often based on only a few species, with other anthozoan calcifiers such as octocorals (soft corals), not being fully considered. We de novo assembled the transcriptomes of four soft-coral species characterized by different calcification strategies (aragonite skeleton vs. calcitic sclerites) and data-mined published nonbilaterian transcriptome resources to construct a taxonomically comprehensive sequence database to map the distribution of scleractinian and octocoral skeletome components. Cnidaria shared no skeletome proteins with Placozoa or Ctenophora, but did share some skeletome proteins with Porifera, such as galaxin-related proteins. Within Scleractinia and Octocorallia, we expanded the distribution for several taxonomically restricted genes such as secreted acidic proteins, scleritin, and carbonic anhydrases, and propose an early, single biomineralization-recruitment event for galaxin sensu stricto. Additionally, we show that the enrichment of acidic residues within skeletogenic proteins did not occur at the Corallimorpharia–Scleractinia transition, but appears to be associated with protein secretion into the organic matrix. Finally, the distribution of octocoral calcification-related proteins appears independent of skeleton mineralogy (i.e., aragonite/calcite) with no differences in the proportion of shared skeletogenic proteins between scleractinians and aragonitic or calcitic octocorals. This points to skeletome homogeneity within but not between groups of calcifying cnidarians, although some proteins such as galaxins and SCRiP-3a could represent instances of commonality.

Octocorallia (class Anthozoa, phylum Cnidaria) is a group of calcifying corals displaying a wide diversity of mineral skeletons. This includes skeletal structures composed of different calcium carbonate polymorphs (aragonite and calcite). This represents a unique feature among anthozoans, as scleractinian corals (subclass Hexacorallia), main reef builders and focus of biomineralization research, are all characterized by an aragonite exoskeleton. From an evolutionary perspective, the presence of aragonitic skeletons in Octocorallia is puzzling as it is observed in very few species and has apparently originated during a Calcite sea (i.e., time interval characterized by calcite-inducing seawater conditions). Despite this, octocorals have been systematically overlooked in biomineralization studies. Here we review what is known about octocoral biomineralization, focusing on the evolutionary and biological processes that underlie calcite and aragonite formation. Although differences in research focus between octocorals and scleractinians are often mentioned, we highlight how strong variability also exists between different octocoral groups. Different main aspects of octocoral biomineralization have been in fact studied in a small set of species, including the (calcitic) gorgonian Leptogorgia virgulata and/or the precious coral Corallium rubrum. These include descriptions of calcifying cells (scleroblasts), calcium transport and chemistry of the calcification fluids. With the exception of few histological observations, no information on these features is available for aragonitic octocorals. Availability of sequencing data is also heterogeneous between groups, with no transcriptome or genome available, for instance, for the clade Calcaxonia. Although calcite represents by far the most common polymorph deposited by octocorals, we argue that studying aragonite-forming could provide insight on octocoral, and more generally anthozoan, biomineralization. First and foremost it would allow to compare calcification processes between octocoral groups, highlighting homologies and differences. Secondly, similarities (exoskeleton) between Heliopora and scleractinian skeletons, would provide further insight on which biomineralization features are driven by skeleton characteristics (shared by scleractinians and aragonitic octocorals) and those driven by taxonomy (shared by octocorals regardless of skeleton polymorph). Including the diversity of anthozoan mineralization strategies into biomineralization studies remains thus essential to comprehensively study how skeletons form and evolved within this ecologically important group of marine animals.

Corals are the ecosystem engineers of coral reefs, one of the most biodiverse marine ecosystems. The ability of corals to form reefs depends on the precipitation of calcium carbonate (CaCO3) under biological control. However, several mechanisms underlying coral biomineralization remain elusive, e.g. whether corals employ different molecular machineries to deposit different CaCO3 polymorphs (i.e., aragonite or calcite). Here we used tandem mass spectrometry (MS/MS) to compare the proteins occluded in the skeleton of three octocoral and one scleractinian species: Tubipora musica and Sinularia cf. cruciata (calcite sclerites), the blue coral Heliopora coerulea (aragonitic skeleton), and the scleractinian aragonitic Montipora digitata. Reciprocal Blast analysis revealed extremely low overlap between aragonitic and calcitic species, while a core set of proteins is shared between octocorals producing calcite sclerites. However, the carbonic anhydrase CruCA4 is present in the skeletons of both polymorphs. Phylogenetic analysis highlighted several possible instances of protein co-option in octocorals. These include acidic proteins and scleritin, which appear to have been secondarily recruited for calcification and likely derive from proteins playing different functions. Similarities between octocorals and scleractinians included presence of a galaxin-related protein, carbonic anhydrases and one hephaestin-like protein. While the first two appear to have been independently recruited, the third appear to share a common origin. This work represents the first attempt to identify and compare proteins associated with coral skeleton polymorph diversity, providing several new research targets and enabling both future functional and evolutionary studies aimed at elucidating the origin and evolution of coral biomineralization.