Single-Cell Genomics Reveals the Divergent Mitochondrial Genomes of Retaria (Foraminifera and Radiolaria).

Mitochondria originated from an ancient bacterial endosymbiont that underwent reductive evolution by gene loss and endosymbiont gene transfer to the nuclear genome. The diversity of mitochondrial genomes published to date has revealed that gene loss and transfer processes are ongoing in many lineages. Most well-studied eukaryotic lineages are represented in mitochondrial genome databases, except for the superphylum Retaria-the lineage comprising Foraminifera and Radiolaria. Using single-cell approaches, we determined two complete mitochondrial genomes of Foraminifera and two nearly complete mitochondrial genomes of radiolarians. We report the complete coding content of an additional 14 foram species. We show that foraminiferan and radiolarian mitochondrial genomes contain a nearly fully overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. In contrast to animals and fungi, many protists encode a diverse set of proteins on their mitochondrial genomes, including several ribosomal genes; however, some aerobic eukaryotic lineages (euglenids, myzozoans, and chlamydomonas-like algae) have reduced mitochondrial gene content and lack all ribosomal genes. Similar to these reduced outliers, we show that retarian mitochondrial genomes lack ribosomal protein and tRNA genes, contain truncated and divergent small and large rRNA genes, and contain only 14 or 15 protein-coding genes, including nad1, -3, -4, -4L, -5, and -7, cob, cox1, -2, and -3, and atp1, -6, and -9, with forams and radiolarians additionally carrying nad2 and nad6, respectively. In radiolarian mitogenomes, a noncanonical genetic code was identified in which all three stop codons encode amino acids. Collectively, these results add to our understanding of mitochondrial genome evolution and fill in one of the last major gaps in mitochondrial sequence databases. IMPORTANCE We present the reduced mitochondrial genomes of Retaria, the rhizarian lineage comprising the phyla Foraminifera and Radiolaria. By applying single-cell genomic approaches, we found that foraminiferan and radiolarian mitochondrial genomes contain an overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. An alternative genetic code was identified in radiolarian mitogenomes in which all three stop codons encode amino acids. Collectively, these results shed light on the divergent nature of the mitochondrial genomes from an ecologically important group, warranting further questions into the biological underpinnings of gene content variability and genetic code variation between mitochondrial genomes.

COI metabarcoding of large benthic Foraminifera: Method validation for application in ecological studies.

Monitoring community composition of Foraminifera (single-celled marine protists) provides valuable insights into environmental conditions in marine ecosystems. Despite the efficiency of environmental DNA (eDNA) and bulk-sample DNA (bulk-DNA) metabarcoding to assess the presence of multiple taxa, this has not been straightforward for Foraminifera partially due to the high genetic variability in widely used ribosomal markers. Here, we test the correctness in retrieving foraminiferal communities by metabarcoding of mock communities, bulk-DNA from coral reef sediment samples, and eDNA from their associated ethanol preservative using the recently sequenced cytochrome c oxidase subunit 1 (COI) marker. To assess the detection success, we compared our results with large benthic foraminiferal communities previously reported from the same sampling sites. Results from our mock communities demonstrate that all species were detected in two mock communities and all but one in the remaining four. Technical replicates were highly similar in number of reads for each assigned ASV in both the mock communities and bulk-DNA samples. Bulk-DNA showed a significantly higher species richness than their associated eDNA samples, and also detected additional species to what was already reported at the specific sites. Our study confirms that metabarcoding using the foraminiferal COI marker adequately retrieves the diversity and community composition of both the mock communities and the bulk-DNA samples. With its decreased variability compared with the commonly used nuclear 18 S rRNA, the COI marker renders bulk-DNA metabarcoding a powerful tool to assess foraminiferal community composition under the condition that the reference database is adequate to the target taxa.

Mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding of Foraminifera communities using taxon-specific primers.

Foraminifera are a species-rich phylum of rhizarian protists that are highly abundant in most marine environments. Molecular methods such as metabarcoding have revealed a high, yet undescribed diversity of Foraminifera. However, so far only one molecular marker, the 18S ribosomal RNA, was available for metabarcoding studies on Foraminifera. Primers that allow amplification of foraminiferal mitochondrial cytochrome oxidase I (COI) and identification of Foraminifera species were recently published. Here we test the performance of these primers for the amplification of whole foraminiferal communities, and compare their performance to that of the highly degenerate LerayXT primers, which amplify the same COI region in a wide range of eukaryotes. We applied metabarcoding to 48 samples taken along three transects spanning a North Sea beach in the Netherlands from dunes to the low tide level, and analysed both sediment samples and meiofauna samples, which contained taxa between 42 µm and 1 mm in body size obtained by decantation from sand samples. We used single-cell metabarcoding (Girard et al., 2022) to generate a COI reference library containing 32 species of Foraminifera, and used this to taxonomically annotate our community metabarcoding data. Our analyses show that the highly degenerate LerayXT primers do not amplify Foraminifera, while the Foraminifera primers are highly Foraminifera- specific, with about 90% of reads assigned to Foraminifera and amplifying taxa from all major groups, i.e., monothalamids, Globothalamea, and Tubothalamea. We identified 176 Foraminifera ASVs and found a change in Foraminifera community composition along the beach transects from high tide to low tide level, and a dominance of single-chambered monothalamid Foraminifera. Our results highlight that COI metabarcoding can be a powerful tool for assessing Foraminiferal communities.

Dynamics of large benthic foraminiferal assemblages: A tool to foreshadow reef degradation?

Ecological regime shifts in the marine realm have been recorded from a variety of systems and locations around the world. Coral reefs have been especially affected, with their benthic habitat changing from a dominance of stony corals to a dominance of other organisms such as fleshy algae. To detect changes in the benthic habitat of coral reefs, simple tools applicable on a global scale are necessary for future monitoring programs. Hence, the aim of this research is to explore the hypothesis that shifts in assemblages of large benthic foraminifera (LBF) can detect early signs of degradation in the reef benthic habitat. To do so, data on living assemblages of LBF collected between 1997 and 2018 at 12 islands in the Spermonde Archipelago (South Sulawesi, Indonesia) were analyzed. Foraminiferal specimens were morphologically identified to the species level and statistical analyses performed to assess changes in their assemblage composition. A clear temporal shift was observed. Typical foraminiferal assemblages in a coral-dominated (e.g., Amphistegina lobifera, Calcarina spengleri, Heterostegina depressa) and fleshy algae-dominated (e.g., Neorotalia gaimardi, C. mayori) reef habitats were identified and significantly linked to the substrate type. Other species (e.g., Elphidium spp., Peneroplis planatus and Sphaerogypsina globulus) seem to reflect a spatial and temporal gradient of anthropogenic pollution from local inhabited islands and ongoing urban development on the mainland. Hence communities of LBF consistently follow gradual shifts in environmental conditions. Additionally to foraminiferal assemblages being an indicator for actual reef condition, closely monitoring LBF may provide early information on reef degradation, in time to take action against identified stressors (e.g., eutrophication or intensive fishing) at local and regional scales. The circumtropical distribution of LBF is such that they can be included worldwide in reef monitoring programs, conditional to calibration to the regional species pool.

First report of mitochondrial COI in foraminifera and implications for DNA barcoding.

Foraminifera are a species-rich phylum of rhizarian protists that are highly abundant in many marine environments and play a major role in global carbon cycling. Species recognition in Foraminifera is mainly based on morphological characters and nuclear 18S ribosomal RNA barcoding. The 18S rRNA contains variable sequence regions that allow for the identification of most foraminiferal species. Still, some species show limited variability, while others contain high levels of intragenomic polymorphisms, thereby complicating species identification. The use of additional, easily obtainable molecular markers other than 18S rRNA will enable more detailed investigation of evolutionary history, population genetics and speciation in Foraminifera. Here we present the first mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences ("barcodes") of Foraminifera. We applied shotgun sequencing to single foraminiferal specimens, assembled COI, and developed primers that allow amplification of COI in a wide range of foraminiferal species. We obtained COI sequences of 49 specimens from 17 species from the orders Rotaliida and Miliolida. Phylogenetic analysis showed that the COI tree is largely congruent with previously published 18S rRNA phylogenies. Furthermore, species delimitation with ASAP and ABGD algorithms showed that foraminiferal species can be identified based on COI barcodes.

Sponges as bioindicators for microparticulate pollutants?

Amongst other threats, the world's oceans are faced with man-made pollution, including an increasing number of microparticulate pollutants. Sponges, aquatic filter-feeding animals, are able to incorporate fine foreign particles, and thus may be a potential bioindicator for microparticulate pollutants. To address this question, 15 coral reef demosponges sampled around Bangka Island (North Sulawesi, Indonesia) were analyzed for the nature of their foreign particle content using traditional histological methods, advanced light microscopy, and Raman spectroscopy. Sampled sponges accumulated and embedded the very fine sediment fraction (<200 µm), absent in the surrounding sand, in the ectosome (outer epithelia) and spongin fibers (skeletal elements), which was confirmed by two-photon microscopy. A total of 34 different particle types were identified, of which degraded man-made products, i.e., polystyrene, particulate cotton, titanium dioxide and blue-pigmented particles, were incorporated by eight specimens at concentrations between 91 and 612 particle/g dry sponge tissue. As sponges can weigh several hundreds of grams, we conservatively extrapolate that sponges can incorporate on average 10,000 microparticulate pollutants in their tissue. The uptake of particles, however, appears independent of the material, which suggests that the fluctuation in material ratios is due to the spatial variation of surrounding microparticles. Therefore, particle-bearing sponges have a strong potential to biomonitor microparticulate pollutants, such as microplastics and other degraded industrial products.

Biodegradation of textile waste by marine bacterial communities enhanced by light.

Knowledge of biofilm formation on pollutants in the marine realm is expanding, but how communities respond to substrates during colonization remains poorly understood. Here, we assess community assembly and respiration in response to two different micropollutants, virgin high-density polyethylene (HDPE) microbeads and textile fibres under different light settings. Raman characterization, high-throughput DNA sequencing data, quantitative PCR, and respiration measurements reveal how a stimulation of aerobic respiration by micropollutants is translated into selection for significantly different communities colonizing the substrates. Despite the lack of evidence for biodegradation of HDPE, an increased abundance and respiration of bacterial taxa closely related to hydrocarbonoclastic Kordiimonas spp. and Alteromonas spp. in the presence of textile waste highlights their biodegradation potential. Incubations with textile fibres exhibited significantly higher respiration rates in the presence of light, which could be partially explained by photochemical dissolution of the textile waste into smaller bioavailable compounds. Our results suggest that the development and increased respiration of these unique microbial communities may potentially play a role in the bioremediation of the relatively long-lived textile pollutants in marine habitats, and that the respiration of heterotrophic hydrocarbon-degrading bacteria colonizing marine pollutants can be stimulated by light.