Spanning over 500 million years, the fossil record of Retaria is a cornerstone of paleobiology, paleoclimate reconstruction, and stratigraphy. Yet, the constitutive branches of Retaria, Foraminifera, and Radiolaria, not only have been studied independently, but just a fraction of this research has focused on living representatives of these lineages. Here we tentatively identify the "Big Five" areas of biological research into Retaria, articulated around novel and transformative understanding of their evolution and diversity, role in ocean biogeochemistry, life cycles, and ecology. Through this perspective, we argue that a unified Retaria framework that seamlessly integrates biological and paleontological data will benefit the scientific community at large, train the next generation of researchers broadly and open novel perspectives to understand the global ocean's evolution.

[This corrects the article DOI: 10.1371/journal.pone.0225246.].

For millions of years, the Mediterranean Sea has regularly experienced episodes of disrupted thermohaline circulation and increased primary productivity that resulted in a largely anoxic water column. These anoxic episodes are typically related to a more humid climate over Northern Africa and are captured in the sedimentary record as organic-rich sapropel layers. Given the excellent preservation of organic molecules in them, sapropels are extraordinary archives for the marine and continental ecosystems associated with the unique conditions that prevailed during their formation. We applied metagenomic environmental DNA (eDNA) analysis to recent sapropels (< 175 kyr) from the Eastern Mediterranean, including Sapropel S5 deposited during the Last Interglacial, and benchmarked obtained results with high resolution geochemical and molecular biomarker records. Ancient eDNA analysis enables reconstructions across all domains of life, including those components of the ecosystem that do not leave fossils or are not recorded in the fossil record. In the case of Mediterranean sapropels, this approach reveals information on both terrestrial and marine ecosystems. We provide detailed insight into vegetation changes in the Nile River Basin during the different, climatically diverse episodes of sapropel deposition. On the marine side, we reveal how water column ecology and major elemental cycles adapted to this massive ecosystem overhaul.

Metabarcoding is a cornerstone of modern ecology, but its accuracy is dependent on the chosen gene marker. While the small subunit ribosomal DNA (SSU) is a powerful tool to describe protist diversity, its reliability in retrieving the composition of communities is less obvious. It is particularly challenging to obtain quantitative estimates of abundance in planktonic foraminifera, where the variability of the SSU gene copy number can span three orders of magnitude. As an alternative, we explored the potential of the mitochondrial cytochrome c oxidase subunit I (COI) marker. We developed a reference barcode library of 130 sequences of a 1200 bp long COI fragment belonging to 26 morphospecies of foraminifera and performed 201 single-cell qPCR quantifications to evaluate the relationship between the number of COI copies, and the size of individual foraminifera. We found that the COI evolves between 25 and 1000 times slower than the SSU and therefore has a poor taxonomic resolution. However, we observed a significant relationship between COI copy number and foraminifera size. These results suggest that SSU and COI can play complementary roles: the SSU is well-suited for capturing taxonomic diversity, while the COI is useful to retrieve crude information on the community composition.

Data on marine microfossil assemblage composition have multiple applications. Initially, they were primarily used for (chrono)stratigraphy and palaeoecology, but these data are now also widely used to study evolutionary and ecological processes, such as past biodiversity and its links with environmental dynamics, or to provide a basis for conservation efforts and biomonitoring. The large range of potential applications renders microfossil abundance data ideal for reuse. However, the complexity inherent in taxonomic data, which encompass extant and extinct species, coupled with the inherent intricacies of information on biological communities extracted from sedimentary archives, poses considerable hurdles in reusing marine microfossil data, even when they are publicly available. Here, we present guidelines derived from an online survey conducted within the marine micropalaeontological community, aimed at improving the reusability of microfossil assemblage data. These guidelines advocate for clarity and transparency in the documentation of the methods and the outcome, and we outline the data attributes required for effective reuse of micropalaeontological data. These guidelines are intended for researchers who generate microfossil abundance datasets and for reviewers, editors, and data curators at repositories. A total of 113 researchers evaluated the relevance of about 50 data attributes that might be needed to enable and maximise the reuse of marine microfossil abundance datasets. Each property is ranked based on the survey results. All information is, in principle, considered “desired”. Information that improves the reusability is ranked as “recommended”, and information that is required for reuse is ranked as “essential”. Analysis of a selection of datasets available online reveals a rather large gap between data properties deemed essential by survey participants and what is actually contained in publicly available microfossil assemblage datasets. While the survey indicates that the micropalaeontological community values good data stewardship, improving data reusability still requires new efforts to incorporate all the essential information. The guidelines presented here are intended as a step in that direction. Determining the optimal forms and formats for data sharing are obvious next steps the community needs to take.

Foraminifera are highly diverse rhizarian protists, with some lineages having developed the ability to retain chloroplasts from algal prey (kleptoplasty). Recently, we revealed the evolutionary relationship between kleptoplasty and algal symbiosis in the benthic foraminifera Hauerina diversa. In this study, we explored fundamental aspects of host-kleptoplast interactions. The photosynthetic rates of H. diversa show the sequestered kleptoplast activity under a wide range of light intensities with no signs of photoinhibition. This lack of photoinhibition response may be attributed to the loss of key elements responsible for this process during the acquisition of kleptoplasts. Our study demonstrates the stability and notably extended retention of kleptoplasty in H. diversa, evidenced by its plastid retention under conditions of heterotrophic feeding deprivation for 50 days. The host-kleptoplast interactions suggest that H. diversa is highly committed to this partnership and that this kleptoplasty species likely relies on similar kleptoplast/alga maintenance mechanisms as symbiont-bearing foraminifera.

Planktonic foraminifera were long considered obligate sexual outbreeders but recent observations have shown that nonspinose species can reproduce by multiple fission. The frequency of multiple fission appears low but the survival rate of the offspring is high and specimens approaching fission can be distinguished. We made use of this observation and established a culturing protocol aimed at enhancing the detection and frequency of fission. Using this protocol, we selectively cultured specimens of Neogloboquadrina pachyderma and raised the frequency of reproduction by fission in culture from 3% in randomly selected specimens to almost 60%. By feeding the resulting offspring different strains of live diatoms, we obtained a thriving offspring population and during the subsequent 6 months of culturing, we observed two more successive generations produced by fission. This provides evidence that in nonspinose species of planktonic foraminifera, reproduction by multiple fission is likely clonal and corresponds to the schizont phase known from benthic foraminifera. We subsequently tested if a similar culturing strategy could be applied to Globigerinita glutinata, representing a different clade of planktonic foraminifera, and we were indeed able to obtain offspring via multiple fission in this species. This work opens new avenues for laboratory-based experimental work with planktonic foraminifera.

Due to ongoing ocean warming, subtropical environments are becoming accessible to tropical species. Among these environments are the vermetid reefs of the Southeastern Mediterranean (SEM). In the last decades, these valuable coastal habitats witnessed the proliferation of numerous alien species of tropical origin. Among the meiofauna thriving on these reefs are benthic foraminifera, single cell marine organisms that make a significant contribution to global carbonate production. It has been widely recognized that benthic foraminifera, among other invasive species, thrive in the macroalgal cover, and it has been suggested that their populations are becoming a significant new source of sediment substrate. Here, we report on the first systematic assessment of the population size of the benthic foraminifera, allowing a comparison with data from the native tropical habitat of these species. Our study is based on a seasonal sampling of benthic foraminifera from confined sampling areas at four sites along the vermetid reef platforms of the Israeli SEM coast. Our survey reveals a patchy distribution of each species with peak population densities exceeding 100,000 specimens per m2, making the SEM a hotspot of benthic foraminifera, with population densities comparable to tropical coral reef environments. The assemblages of the SEM hotspot are dominated by cosmopolitan foraminiferal taxa and tropical invaders from the Indo-Pacific (e.g., Amphistegina lobifera, Pararotalia calcariformata, soritids, and Hauerina diversa). In contrast to foraminiferal hotspots in the tropics, which are completely dominated by larger symbiont-bearing taxa, the SEM hotspot stands out due to high abundances of non-symbiont-bearing species Textularia agglutinans and small miliolids. An intriguing observation is the significant heterogeneity in composition and density of foraminiferal assemblages between the vermetid reefs' southern and northern areas (Israel), indicating that the productivity of the dominant species are also modulated by local yet unknown environmental factors.

Visual as well as genetic biometrics are routinely employed to identify species and individuals in biological applications. However, no attempts have been made in this domain to computationally enhance visual classification of rare classes with little image data via genetics. In this paper, we thus propose aligned visual-genetic learning as a new application domain with the aim to implicitly encode cross-modality associations for improved performance. We demonstrate for the first time that such alignment can be achieved via deep embedding models and that the approach is directly applicable to boosting long-tailed recognition (LTR), particularly for rare species. We experimentally demonstrate the efficacy of the concept via application to microscopic imagery of 30k+ planktic foraminifer shells across 32 species when used together with independent genetic data samples. Most importantly for practitioners, we show that visual-genetic alignment can significantly benefit visual-only recognition of the rarest species. Technically, we pre-train a visual ResNet50 deep learning model using triplet loss formulations to create an initial embedding space. We re-structure this space based on genetic anchors embedded via a Sequence Graph Transform (SGT) and linked to visual data by cross-domain cosine alignment. We show that an LTR approach improves the state-of-the-art across all benchmarks and that adding our visual-genetic alignment improves per-class and particularly rare tail class benchmarks significantly further. Overall, visual-genetic LTR training raises rare per-class accuracy from 37.4% to benchmark-beating 59.7%. We conclude that visual-genetic alignment can be a highly effective tool for complementing visual biological data containing rare classes. The concept proposed may serve as an important future tool for integrating genetics and imageomics towards a more complete scientific representation of taxonomic spaces and life itself. Code, weights, and data splits are published for full reproducibility.

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The nature and extent of diversity in the plankton has fascinated scientists for over a century. Initially, the discovery of many new species in the remarkably uniform and unstructured pelagic environment appeared to challenge the concept of ecological niches. Later, it became obvious that only a fraction of plankton diversity had been formally described, because plankton assemblages are dominated by understudied eukaryotic lineages with small size that lack clearly distinguishable morphological features. The high diversity of the plankton has been confirmed by comprehensive metabarcoding surveys, but interpretation of the underlying molecular taxonomies is hindered by insufficient integration of genetic diversity with morphological taxonomy and ecological observations. Here we use planktonic foraminifera as a study model and reveal the full extent of their genetic diversity and investigate geographical and ecological patterns in their distribution. To this end, we assembled a global data set of ~7600 ribosomal DNA sequences obtained from morphologically characterised individual foraminifera, established a robust molecular taxonomic framework for the observed diversity, and used it to query a global metabarcoding data set covering ~1700 samples with ~2.48 billion reads. This allowed us to extract and assign 1 million reads, enabling characterisation of the structure of the genetic diversity of the group across ~1100 oceanic stations worldwide. Our sampling revealed the existence of, at most, 94 distinct molecular operational taxonomic units (MOTUs) at a level of divergence indicative of biological species. The genetic diversity only doubles the number of formally described species identified by morphological features. Furthermore, we observed that the allocation of genetic diversity to morphospecies is uneven. Only 16 morphospecies disguise evolutionarily significant genetic diversity, and the proportion of morphospecies that show genetic diversity increases poleward. Finally, we observe that MOTUs have a narrower geographic distribution than morphospecies and that in some cases the MOTUs belonging to the same morphospecies (cryptic species) have different environmental preferences. Overall, our analysis reveals that even in the light of global genetic sampling, planktonic foraminifera diversity is modest and finite. However, the extent and structure of the cryptic diversity reveals that genetic diversification is decoupled from morphological diversification, hinting at different mechanisms acting at different levels of divergence.

Describing living community compositions is essential to monitor ecosystems in a rapidly changing world, but it is challenging to produce fast and accurate depiction of ecosystems due to methodological limitations. Morphological methods provide absolute abundances with limited throughput, whereas metabarcoding provides relative abundances of genes that may not correctly represent living communities from environmental DNA assessed with morphological methods. However, it has the potential to deliver fast descriptions of living communities provided that it is interpreted with validated species-specific calibrations and reference databases. Here, we developed a quantitative approach to retrieve from metabarcoding data the assemblages of living large benthic foraminifera (LBF), photosymbiotic calcifying protists, from Indonesian coral reefs that are under increasing anthropogenic pressure. To depict the diversity, we calculated taxon-specific correction factors to reduce biological biases by comparing surface area, biovolume and calcite volume, and the number of mitochondrial gene copies in seven common LBF species. To validate the approach, we compared calibrated datasets of morphological communities from mock samples with bulk reef sediment; both sample types were metabarcoded. The calibration of the data significantly improved the estimations of genus relative abundance, with a difference of +/- 5% on average, allowing for comparison of past morphological datasets with future molecular ones. Our results also highlight the application of our quantitative approach to support reef monitoring operations by capturing fine-scale processes, such as seasonal and pollution-driven dynamics, that require high-throughput sampling treatment.

Calcite shells of planktic foraminifera (Protista, Rhizaria) constitute a large portion of deep-sea sediments. The shells are constructed by sequential addition of partly overlapping chambers with diverse shapes, resulting in complex shell architectures, which are genetically fixed and diagnostic at the species level. The characterisation of the complete architecture requires three-dimensional imaging of the shell, including the partially or entirely covered juvenile chambers. Here we provide reconstructed x-ray micro computed tomography image stacks of 179 specimens of extant planktic foraminifera collected from plankton tows, sediment traps and surface sediments. The specimens have fully resolved and curated taxonomy and represent 43 of the currently recognised 48 holoplanktic species and subspecies. The image stacks form a basis for further applications, such as the characterisation of the architectural morphospace of the extant taxa, allowing studies of species functional ecology, calcification intensity and reconstructions of phylogenetic relationships.

Foraminifera are unicellular organisms that established the most diverse algal symbioses in the marine realm. Endosymbiosis repeatedly evolved in several lineages, while some engaged in the sequestration of chloroplasts, known as kleptoplasty. So far, kleptoplasty has been documented exclusively in the rotaliid clade. Here, we report the discovery of kleptoplasty in the species Hauerina diversa that belongs to the miliolid clade. The existence of kleptoplasty in the two main clades suggests that it is more widespread than previously documented. We observed chloroplasts in clustered structures within the foraminiferal cytoplasm and confirmed their functionality. Phylogenetic analysis of 18S ribosomal RNA gene sequences showed that H. diversa branches next to symbiont-bearing Alveolinidae. This finding represents evidence of of a relationship between kleptoplastic and symbiotic foraminifera.. Analysis of ribosomal genes and metagenomics revealed that alveolinid symbionts and kleptoplasts belong to the same clade, which suggests a common ancestry.

The reproductive strategy of planktonic foraminifera, key pelagic calcifiers, has long remained elusive, hampering efforts to understand and model their population dynamics. This is particularly critical in polar oceans where their success relies on rapid population growth after the polar night. Here, we provide field and laboratory observations constraining the reproductive cycle of the dominant polar species Neogloboquadrina pachyderma in Antarctic and Arctic waters. We observe that asexual reproduction is a rare but persistent element of the reproductive strategy and that it is reflected in a dimorphism in proloculus and in the ratio of shell size versus the number of chambers between sexually and asexually produced individuals. This dimorphism in natural populations reveals that asexual reproduction supplies more than 75% of adult individuals. This indicates a multigenerational reproduction strategy, where the majority of the population releases gametes to facilitate recombination, while a minority persistently reproduces asexually, allowing rapid population growth as a prerequisite for success in the polar oceans.

Among the most successful Lessepsian invaders is the symbiont-bearing benthic foraminifera Amphistegina lobifera. In its newly conquered habitat, this prolific calcifier and ecosystem engineer is exposed to environmental conditions that exceed the range of its native habitat. To disentangle which processes facilitated the invasion success of A. lobifera into the Mediterranean Sea we analyzed a ~ 1400 bp sequence fragment covering the SSU and ITS gene markers to compare the populations from its native regions and along the invasion gradient. The genetic variability was studied at four levels: intra-genomic, population, regional and geographical. We observed that the invasion is not associated with genetic differentiation, but the invasive populations show a distinct suppression of intra-genomic variability among the multiple copies of the rRNA gene. A reduced genetic diversity compared to the Indopacific is observed already in the Red Sea populations and their high dispersal potential into the Mediterranean appears consistent with a bridgehead effect resulting from the postglacial expansion from the Indian Ocean into the Red Sea. We conclude that the genetic structure of the invasive populations reflects two processes: high dispersal ability of the Red Sea source population pre-adapted to Mediterranean conditions and a likely suppression of sexual reproduction in the invader. This discovery provides a new perspective on the cost of invasion in marine protists: The success of the invasive A. lobifera in the Mediterranean Sea comes at the cost of abandonment of sexual reproduction.

Ribosomal intragenomic variability in prokaryotes and eukaryotes is a genomic feature commonly studied for its inflationary impact on molecular diversity assessments. However, the evolutionary mechanisms and distribution of this phenomenon within a microbial group are rarely explored. Here, we investigate the intragenomic variability in 33 species of planktonic foraminifera, calcifying marine protists, by inspecting 2,403 partial SSU sequences obtained from single-cell clone libraries. Our analyses show that polymorphisms are common among planktonic foraminifera species, but the number of polymorphic sites significantly differs among clades. With our molecular simulations, we could assess that most of these mutations are located in paired regions that do not affect the secondary structure of the SSU fragment. Finally, by mapping the number of polymorphic sites on the phylogeny of the clades, we were able to discuss the evolution and potential sources of intragenomic variability in planktonic foraminifera, linking this trait to the distinctive nuclear and genomic dynamics of this microbial group.

Shallow-water banks of foraminiferal propagules (tiny juveniles <63 µm) harbor a hidden assemblage composition that has altered our understanding of traditional distribution patterns. Propagules can be transported well beyond their environmental limits and remain dormant until local conditions become suitable. Therefore, the composition of propagule assemblages is expected to differ from “adult” assemblages. This offers various opportunities for a better understanding of foraminiferal reactions to environmental changes and also of the variability of living foraminiferal populations throughout the year. To date, propagule assemblages have only been analyzed “passively” through growth experiments with propagule banks under laboratory conditions. Here, we apply for the first time a combination of a multi-week cultivation experiment and eDNA metabarcoding of the different size fractions. Sediment samples were taken from a shallow lagoon in Corfu (Greece) and sieved over 63 µm to separate the propagule bank from the coarser fraction. The in-situ material of the sampling site was used as a baseline for the subsequent experiment (T0). The finer fraction (<63 µm) was set up in a culture experiment for 15 weeks under stable conditions (22°C, 38 psu, constant aeration). The cultures were repeatedly harvested for grown foraminifera (>63 µm) every 5 weeks (T1–T3). At the same intervals, samples were taken and re-sieved over 63 µm for eDNA metabarcoding of both size fractions. The morphology-based count data of the foraminiferal specimens revealed significantly different assemblage compositions after each harvest (T0–T3). The differences between the in-situ (T0) and the experimental samples (T1–T3) were most distinct, indicating a specific composition of the propagule bank. The differences between in-situ and experimental assemblages as well as the shifting assemblage compositions over time were mirrored by the metabarcoding data from the respective intervals. Our results highlight the potential for metabarcoding to complement and expand insights gained from morphology-based approaches in foraminiferal studies.

Remote deep-ocean sediment (DOS) ecosystems are among the least explored biomes on Earth. Genomic assessments of their biodiversity have failed to separate indigenous benthic organisms from sinking plankton. Here, we compare global-scale eukaryotic DNA metabarcoding datasets (18S-V9) from abyssal and lower bathyal surficial sediments and euphotic and aphotic ocean pelagic layers to distinguish plankton from benthic diversity in sediment material. Based on 1685 samples collected throughout the world ocean, we show that DOS diversity is at least threefold that in pelagic realms, with nearly two-thirds represented by abundant yet unknown eukaryotes. These benthic communities are spatially structured by ocean basins and particulate organic carbon (POC) flux from the upper ocean. Plankton DNA reaching the DOS originates from abundant species, with maximal deposition at high latitudes. Its seafloor DNA signature predicts variations in POC export from the surface and reveals previously overlooked taxa that may drive the biological carbon pump.