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ABSTRACT 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.

Abstract 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.

Abstract 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.

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.

Traditional morphological methods for species identification are highly time consuming, especially for small organisms, such as Foraminifera, a group of shell-building microbial eukaryotes. To analyze large amounts of samples more efficiently, species identification methods have extended to molecular tools in the last few decades. Although a wide range of phyla have good markers available, for Foraminifera only one hypervariable marker from the ribosomal region (18S) is widely used. Recently a new mitochondrial marker cytochrome oxidase subunit 1 (COI) has been sequenced. Here we investigate whether this marker has a higher potential for species identification compared to the ribosomal marker. We explore the genetic variability of both the 18S and COI markers in 22 benthic foraminiferal morphospecies (orders Miliolida and Rotaliida). Using single-cell DNA, the genetic variability within specimens (intra) and between specimens (inter) of each species was assessed using next-generation sequencing. Amplification success rate was twice as high for COI (151/200 specimens) than for 18S (73/200 specimens). The COI marker showed greatly decreased intra- and inter-specimen variability compared to 18S in six out of seven selected species. The 18S phylogenetic reconstruction fails to adequately cluster multiple species together in contrast to COI. Additionally, the COI marker helped recognize misclassified specimens difficult to morphologically identify to the species level. Integrative taxonomy, combining morphological and molecular characteristics, provides a robust picture of the foraminiferal species diversity. Finally, we suggest the use of a set of sequences (two or more) to describe species showing intra-genomic variability additionally to using multiple markers. Our findings highlight the potential of the newly discovered mitochondrial marker for molecular species identification and metabarcoding purposes.