ForametCeTera, a novel CT scan dataset to expedite classification research of (non-)foraminifera.

This paper introduces ForametCeTera, a pioneering dataset designed to address the challenges associated with automating the analysis of benthic foraminifera in sediment cores. Foraminifera are sensitive sentinels of environmental change and are a crucial component of carbonate-denominated ecosystems, such as coral reefs. Studying their prevalence and characteristics is imperative in understanding climate change. However, analysis of foraminifera contained in core samples currently requires washing, sieving and manual quantification. These methods are thus time-consuming and require trained experts. To overcome these limitations, we propose an alternative workflow utilizing 3D X-ray computational tomography (CT) for fully automated analysis, saving time and resources. Despite recent advancements in automation, a crucial lack of methods persists for segmenting and classifying individual foraminifera from 3D scans. In response, we present ForametCeTera, a diverse dataset featuring 436 3D CT scans of individual foraminifera and non-foraminiferan material following a high-throughput scanning workflow. ForametCeTera serves as a foundational resource for generating synthetic digital core samples, facilitating the development of segmentation and classification methods of entire core sample CT scans.

Single chamber Mg/Ca analyses of Globigerinoides ruber for paleo-proxy calibration using femtosecond LA-ICP-MS.

Mg/Ca is an independent proxy in paleoceanography to reconstruct past seawater temperature. Femtosecond Laser Ablation Inductively Coupled Plasma Mass Spectrometry (fs-LA-ICP-MS) was employed to determine the Mg/Ca composition of tests (shells) of the planktic foraminifer species Globigerinoides ruber albus (white chromotype) and G. ruber ruber (red/pink chromotype) sampled alive from the temperate to subtropical eastern North Atlantic with the research sailing yacht Eugen Seibold. Mg/Ca data are compared to (i) the measured in-situ temperature of ambient seawater, (ii) average mixed layer temperature, and (iii) sea surface temperature (SST). The pooled mean chamber Mg/Ca from each plankton tow site exhibits a positive relationship with SST. Two chamber-specific calibrations are derived, which are consistent with previous calibration equations for comparable paleo-archives. The results confirm fs-LA-ICP-MS as reliable method for determining Mg/Ca in G. ruber, and both the penultimate and antepenultimate chambers of adult specimens may provide comprehensible Mg/Ca temperatures of the surface ocean.

Measuring ecological quality status in low-diversity Arctic intertidal foraminiferal assemblages using a diversity-based index.

EcoQS assessment of the marine intertidal zone based on its fauna is challenging because the assemblages have a low diversity and consist of stress tolerant species. The new approach we propose is to pool foraminiferal diversity (effective number of species exp(H'bc)) across the whole intertidal zone including the salt marsh and tidal flat. In seven fjordheads studied in northern Fennoscandia, polycyclic aromatic hydrocarbon (PAH) concentrations indicated low levels of pollution (EcoQSPAH Excellent to Moderate). Jadammina or Balticammina dominated the salt marsh, Elphidium albiumbilicatum, Elphidium williamsoni, Elphidium clavatum, and Buccella frigida occurred in the tidal flat. Ovammina opaca thrived in both belts. While foraminiferal test abnormalities are often proposed to measure pollution impacts, we did not detect any correlation with PAHs. EcoQS based on foraminiferal diversity (EcoQSforam Excellent to Good) matched EcoQS based on PAHs suggesting that pooled foraminiferal diversity reliably measures intertidal EcoQS.

Sediment DNA metabarcoding and morphology provide complementary insight into macrofauna and meiobenthos response to environmental gradients in an Arctic glacial fjord.

Arctic fjords ecosystems are highly dynamic, with organisms exposed to various natural stressors along with productivity clines driven by advection of water masses from shelves. The benthic response to these environmental clines has been extensively studied using traditional, morphology-based approaches mostly focusing on macroinvertebrates. In this study we analyse the effects of glacially mediated disturbance on the biodiversity of benthic macrofauna and meiobenthos (meiofauna and Foraminifera) in a Svalbard fjord by comparing morphology and eDNA metabarcoding. Three genetic markers targeting metazoans (COI), meiofauna (18S V1V2) and Foraminifera (18S 37f) were analyzed. Univariate measures of alpha diversity and multivariate compositional dissimilarities were calculated and tested for similarities in response to environmental gradients using correlation analysis. Our study showed different taxonomic composition of morphological and molecular datasets for both macrofauna and meiobenthos. Some taxonomic groups while abundant in metabarcoding data were almost absent in morphology-based inventory and vice versa. In general, species richness and diversity measures in macrofauna morphological data were higher than in metabarcoding, and similar for the meiofauna. Both methodological approaches showed different patterns of response to the glacially mediated disturbance for the macrofauna and the meiobenthos. Macrofauna showed an evident distinction in taxonomic composition and a dramatic cline in alpha diversity indices between the outer and inner parts of fjord, while the meiobenthos showed a gradual change and more subtle responses to environmental changes along the fjord axis. The two methods can be seen as complementing rather than replacing each other. Morphological approach provides more accurate inventory of larger size species and more reliable quantitative data, while metabarcoding allows identification of inconspicuous taxa that are overlooked in morphology-based studies. As different taxa may show different sensitivities to environmental changes, both methods shall be used to monitor marine biodiversity in Arctic ecosystems and its response to dramatically changing environmental conditions.

Effect of micro-plastic particles on coral reef foraminifera.

Foraminifera are single-celled protists which are important mediators of the marine carbon cycle. In our study, we explored the potential impact of polystyrene (PS) microplastic particles on two symbiont-bearing large benthic foraminifera species-Heterostegina depressa and Amphistegina lobifera-over a period of three weeks, employing three different approaches: investigating (1) stable isotope (SI) incorporation-via 13C- and 15N-labelled substrates-of the foraminifera to assess their metabolic activity, (2) photosynthetic efficiency of the symbiotic diatoms using imaging PAM fluorometry, and (3) microscopic enumeration of accumulation of PS microplastic particles inside the foraminiferal test. The active feeder A. lobifera incorporated significantly more PS particles inside the cytoplasm than the non-feeding H. depressa, the latter accumulating the beads on the test surface. Photosynthetic area of the symbionts tended to decrease in the presence of microplastic particles in both species, suggesting that the foraminiferal host cells started to digest their diatom symbionts. Compared to the control, the presence of microplastic particles lead to reduced SI uptake in A. lobifera, which indicates inhibition of inorganic carbon and nitrogen assimilation. Competition for particulate food uptake was demonstrated between algae and microplastic particles of similar size. Based on our results, both species seem to be sensitive to microplastic pollution, with non-feeding H. depressa being more strongly affected.

Exploring the underwater life in transitional environments: Benthic foraminifera, ostracods, and dinoflagellate cysts - Biotic trends and EcoQS assessment in the Mar Piccolo of Taranto (Ionian sea, southern Italy).

Coastal areas have historically thrived as centers of human activities due to their resources, economic opportunities, and natural allure. The rapid growth of coastal populations has however brought forth a multitude of challenges to tackle, with pollution emerging as a significant and far-reaching issue. Our study focuses on the Mar Piccolo of Taranto (Ionian Sea, Southern Italy), a lagoon-like coastal basin (separated in two sub-basins) that, since decades, has been heavily affected by human activities and aquaculture, leading to environmental deterioration. Although past studies have looked at environmental conditions in the Mar Piccolo from a chemical perspective, the biological component (e.g., biological indicators) has been mostly neglected. In this study, we firstly aim to examine the distribution and diversity of foraminifera, ostracods, and dinoflagellate cysts in December 2016 and compare our findings with data collected in December 2011. Foraminiferal and ostracod communities exhibit similar patterns in the two sampling campaigns, while the communities of encysted dinoflagellates show differences concerning both densities and diversity. Then, we evaluate the Ecological Quality Status (EcoQS) using ecological indices. While the indices in the inner basin appear to reflect an actual ecological degradation, they yield conflicting results in the outer basin. In the outer basin, indeed, the indices overestimate the EcoQS. This study highlights the potential of these indices for characterizing the EcoQS but emphasizes the need for improvements in their reliability. This research also contributes to a more holistic understanding of environmental condition in the Mar Piccolo and underscores the importance of integrating biological quality elements into ecosystem management and remediation strategies.

ForCenS-LGM: a dataset of planktonic foraminifera species assemblage composition for the Last Glacial Maximum.

Species assemblage composition of marine microfossils offers the possibility to investigate ecological and climatological change on time scales inaccessible using conventional observations. Planktonic foraminifera - calcareous zooplankton - have an excellent fossil record and are used extensively in palaeoecology and palaeoceanography. During the Last Glacial Maximum (LGM; 19,000 - 23,000 years ago), the climate was in a radically different state. This period is therefore a key target to investigate climate and biodiversity under different conditions than today. Studying LGM climate and ecosystems indeed has a long history, yet the most recent global synthesis of planktonic foraminifera assemblage composition is now nearly two decades old. Here we present the ForCenS-LGM dataset with 2,365 species assemblage samples collected using standardised methods and with harmonised taxonomy. The data originate from marine sediments from 664 sites and present a more than 50% increase in coverage compared to previous work. The taxonomy is compatible with the most recent global core top dataset, enabling direct investigation of temporal changes in foraminifera biogeography and facilitating seawater temperature reconstructions.

Biogeographic response of marine plankton to Cenozoic environmental changes.

In palaeontological studies, groups with consistent ecological and morphological traits across a clade's history (functional groups)1 afford different perspectives on biodiversity dynamics than do species and genera2,3, which are evolutionarily ephemeral. Here we analyse Triton, a global dataset of Cenozoic macroperforate planktonic foraminiferal occurrences4, to contextualize changes in latitudinal equitability gradients1, functional diversity, palaeolatitudinal specialization and community equitability. We identify: global morphological communities becoming less specialized preceding the richness increase after the Cretaceous-Palaeogene extinction; ecological specialization during the Early Eocene Climatic Optimum, suggesting inhibitive equatorial temperatures during the peak of the Cenozoic hothouse; increased specialization due to circulation changes across the Eocene-Oligocene transition, preceding the loss of morphological diversity; changes in morphological specialization and richness about 19 million years ago, coeval with pelagic shark extinctions5; delayed onset of changing functional group richness and specialization between hemispheres during the mid-Miocene plankton diversification. The detailed nature of the Triton dataset permits a unique spatiotemporal view of Cenozoic pelagic macroevolution, in which global biogeographic responses of functional communities and richness are decoupled during Cenozoic climate events. The global response of functional groups to similar abiotic selection pressures may depend on the background climatic state (greenhouse or icehouse) to which a group is adapted.

Benthic foraminiferal community structure and its response to environmental factors revealed using high-throughput sequencing in the Zhoushan Fishing Ground, East China Sea.

Benthic foraminifera are excellent tools for monitoring marine environments and reconstructing paleoenvironments. This study investigated the structure and diversity of benthic foraminiferal communities in 20 superficial sediment samples obtained from the Zhoushan Fishing Ground (ZFG) using high-throughput sequencing based on small subunit ribosomal DNA and RNA amplification. The results revealed Rotaliida as the most dominant group, with spatial heterogeneity in foraminiferal distribution. Total benthic foraminiferal communities exhibited higher species richness and diversity compared to active communities. While heavy metal pollution in the ZFG was moderate, areas with elevated concentrations of heavy metals exhibited low diversity and richness in foraminiferal communities. Total foraminiferal community structure was primarily influenced by factors such as water depth and Hg, Pb, Cd, and Zn levels. Notably, Hg levels emerged as a critical factor impacting the structure and diversity of the active foraminiferal community. The dominant species, Operculina, exhibited tolerance toward heavy metal pollution.

Rapid Freezing and Cryo-SEM-EDS Imaging of Foraminifera (Unicellular Eukaryotes) Using a Conductive Viscous Cryogenic Glue.

Spatial distribution of water-soluble molecules and ions in living organisms is still challenging to assess. Energy-dispersive X-ray spectroscopy (EDS) via cryogenic scanning electron microscopy (cryo-SEM) is one of the promising methods to study them without loss of dissolved contents. High-resolution cryo-SEM-EDS has challenges in sample preparation, including cross-section exposure and sample drift/charging due to insulative surrounding water. The former becomes problematic for large and inseparable organisms, such as benthic foraminifera, a unicellular eukaryote playing significant roles in marine ecosystems, which often exceed the size limit for the most reliable high-pressure freezing. Here we show graphite oxide dispersed in sucrose solution as a good glue to freeze, expose cross-section by cryo-ultramicrotome, and analyze elemental distribution owing to the glue's high viscosity, adhesion force, and electron conductivity. To demonstrate the effectiveness and applicability of the glue for cryo-SEM-EDS, deep-sea foraminifer Uvigerina akitaensis was sampled during a cruise and plunge frozen directly on the research vessel, where the liquid nitrogen supply is limited. The microstructures were preserved as faithfully in cryo-SEM images as those with the conventional resin-substituted transmission electron micrograph. We found elements colocalized within the cytoplasm originating from water-soluble compounds that can be lost with conventional dehydrative fixation.

New xenophyophores (Foraminifera, Monothalamea) from the eastern Clarion-Clipperton Zone (equatorial Pacific).

Xenophyophores are large, agglutinated foraminifera that dominate the benthic megafauna in some parts of the deep sea. Here, we describe an assemblage of largely fragmentary specimens from the Clarion-Clipperton Zone (CCZ), an area of the eastern abyssal Pacific hosting large, commercially significant deposits of polymetallic nodules. We recognised 18 morphospecies of which eight yielded DNA sequences. These include two new genera and three new species, Claraclippia seminuda gen. & sp. nov., Stereodiktyoma mollis gen. & sp. nov., and Aschemonella tani sp. nov., three that are assigned to known species, Abyssalia foliformis, Aschemonella monilis and Shinkaiya contorta, and two assigned to open nomenclature forms Abyssalia aff. foliformis and Stannophyllum aff. granularium. An additional ten forms are represented only by morphology. The following seven are placed in known genera, species and open-nomenclature forms: Aschemonella? sp., Homogammina sp., Psammina multiloculata, P. aff. multiloculata, P. aff. limbata form 1 sensu Gooday et al., 2018, P. aff. limbata form 2 sensu Gooday et al., 2018, and Stannophyllum spp. The other three could not be identified to genus level. This new collection brings the total of described and undescribed species and morphotypes from the CCZ to 27 and 70, respectively, reinforcing the already high diversity of xenophyophores known from this part of the Pacific.

Impact of pesticides on marine coral reef foraminifera.

Our laboratory study looked into how pesticides affect the foraminifera species Heterostegina depressa and their obligatory algal endosymbionts. We incubated the foraminifera separately with different types of pesticides at varying concentrations (1 %, 0.01 % and 0.0001 %); we included the insecticide Confidor© (active substance: imidacloprid), the fungicide Pronto©Plus (tebuconazole), and the herbicide Roundup© (glyphosate). Our evaluation focused on the symbiont's photosynthetically active area (PA), and the uptake of dissolved inorganic carbon (DIC) and nitrogen (nitrate) to determine the vitality of the foraminifera. Our findings showed that even the lowest doses of the fungicide and herbicide caused irreparable damage to the foraminifera and their symbionts. While the insecticide only deactivated the symbionts (PA = 0) at the highest concentration (1 %), the fungicide, and herbicide caused complete deactivation even at the lowest levels provided (0.0001 %). The fungicide had the strongest toxic effect on the foraminiferal host regarding reduced isotope uptake. In conclusion, all pesticides had a negative impact on the holosymbiont, with the host showing varying degrees of sensitivity towards different types of pesticides.

A matter of choice: Understanding the interactions between epiphytic foraminifera and their seagrass host Halophila stipulacea.

In sub/tropical waters, benthic foraminifera are among the most abundant epiphytic organisms inhabiting seagrass meadows. This study explored the nature of the association between foraminifera and the tropical seagrass species H. stipulacea, aiming to determine whether these interactions are facilitative or random. For this, we performed a "choice" experiment, where foraminifera could colonize H. stipulacea plants or plastic "seagrasses" plants. At the end of the experiment, a microbiome analysis was performed to identify possible variances in the microbial community and diversity of the substrates. Results show that foraminifera prefer to colonize H. stipulacea, which had a higher abundance and diversity of foraminifera than plastic seagrass plants, which increased over time and with shoot age. Moreover, H. stipulacea leaves have higher epiphytic microbial community abundance and diversity. These results demonstrate that seagrass meadows are important hosts of the foraminifera community and suggest the potential facilitative effect of H. stipulacea on epiphytic foraminifera, which might be attributed to a greater diversity of the microbial community inhabiting H. stipulacea.

Intrusion of Arabian Sea high salinity water and monsoon-associated processes modulate planktic foraminiferal abundance and carbon burial in the southwestern Bay of Bengal.

The unicellular calcareous planktic foraminifera sequester a significant portion of the carbon dioxide dissolved in the ocean, thus burying the carbon in sediments for millions of years. The global warming and associated processes are likely to affect the planktic foraminiferal abundance and diversity. Therefore, their baseline distribution has to be documented and correlated with ambient parameters to assess its fate under different climate change scenarios. Here, we report an exceptionally high abundance of planktic foraminifera and thus large carbon burial in the southwestern Bay of Bengal. The very high absolute abundance of planktic foraminifera in the Cauvery River basin is attributed to biannual productivity, warmer and saline waters. Globigerinita glutinata is the highest abundant species followed by Globigerinoides ruber and Globigerina bulloides. Globigerina bulloides is abundant on the shelf, where the upwelling is more frequent. The relative abundance of Globorotalia menardii is positively correlated with thermocline salinity and negatively correlated with thermocline temperature. Similarly, Neogloboquadrina dutertrei and Globoquadrina conglomerata are negatively correlated with mixed layer as well as thermocline temperature and mixed layer salinity. Both these species are positively correlated with thermocline salinity. Globigerina falconensis is more abundant in the southernmost transect influenced by intense winter monsoon precipitation. We report that G. ruber prefers high saline and warmer waters with the highest abundance in the southernmost transect. From the foraminiferal distribution, it is evident that the temperature and salinity of the mixed layer as well as thermocline, food availability, and monsoon-associated processes affect the planktic foraminiferal abundance and thus carbon burial in the southwestern Bay of Bengal. The changes in influx of southeastern Arabian Sea water will affect the planktic foraminiferal population and subsequent carbon burial in the southwestern Bay of Bengal.

Benthic foraminifera in Gulf of Mexico show temporal and spatial dynamics of microplastics.

Microplastics have accumulated in the environment since plastic production began, with present-day observations that range from marine trenches to mountains. However, research on microplastics has only recently begun so it is unclear how they have changed over time in many oceanic regions. Our study addressed this gap by quantifying the temporal and spatial dynamics of microplastics in two deep-water regions of the Gulf of Mexico (GOM). We isolated agglutinated foraminifera from sediment cores and assessed microplastics that were incorporated into their tests. Our results indicated that microplastics were incorporated by agglutinated foraminifera after plastic production began. Microplastics were higher at deep-water sites and closer to the Mississippi River. This study confirms the presence of microplastic incorporation into agglutinated foraminifera tests and investigates microplastics in deep-water sediments in the GOM. Additional work is needed to fully identify the distribution of microplastics across the GOM and other oceanic basins.

Deciphering the impact of decabromodiphenyl ether (BDE-209) on benthic foraminiferal communities: Insights from Cell-Tracker Green staining and eDNA metabarcoding.

This study investigates the ecotoxicological effects of BDE-209, a persistent organic pollutant (POP) prevalent in Kuwait's coastal-industrial areas, on benthic foraminiferal communities. We conducted a mesocosm experiment in which we exposed benthic foraminiferal communities sampled from the coastal-industrial areas of Kuwait to a gradient of BDE-209 concentrations (0.01 to 20 mg/kg). The impact of exposure was assessed using live-staining and metabarcoding techniques. Despite the significantly different taxonomic compositions detected by the two techniques, our results show that BDE-209 significantly affects foraminiferal communities, with moderately high concentrations leading to reduced α-diversity and considerable taxonomic shifts in both molecular and morphological assemblages. At concentrations of 10 and 20 mg/kg, no living foraminifera were detected after 8 weeks, suggesting a threshold for their survival under BDE-209 exposure. The parallel responses of molecular and morphological communities confirm the reliability of both assessment methods. This study is the first to investigate the reaction of eukaryotic communities, specifically foraminifera, to POPs such as BDE-209, generating valuable insights that have the potential to enhance field studies and aid the refinement of sediment quality guidelines.

Foraminifera as a model of eukaryotic genome dynamism.

In contrast to the canonical view that genomes cycle only between haploid and diploid states, many eukaryotes have dynamic genomes that change content throughout an individual's life cycle. However, the few detailed studies of microeukaryotic life cycles render our understanding of eukaryotic genome dynamism incomplete. Foraminifera (Rhizaria) are an ecologically important, yet understudied, clade of microbial eukaryotes with complex life cycles that include changes in ploidy and genome organization. Here, we apply fluorescence microscopy and image analysis techniques to over 2,800 nuclei in 110 cells to characterize the life cycle of Allogromia laticollaris strain Cold Spring Harbor (CSH), one of few cultivable foraminifera species. We show that haploidy and diploidy are brief moments in the A. laticollaris life cycle and that A. laticollaris nuclei endoreplicate up to 12,000 times the haploid genome size. We find that A. laticollaris reorganizes a highly endoreplicated nucleus into thousands of haploid genomes through a non-canonical mechanism called Zerfall, in which the nuclear envelope degrades and extrudes chromatin into the cytoplasm. Based on these findings, along with changes in nuclear architecture across the life cycle, we believe that A. laticollaris uses spatio-temporal mechanisms to delineate germline and somatic DNA within a single nucleus. The analyses here extend our understanding of the genome dynamics across the eukaryotic tree of life.IMPORTANCEIn traditional depictions of eukaryotes (i.e., cells with nuclei), life cycles alternate only between haploid and diploid phases, overlooking studies of diverse microeukaryotic lineages (e.g., amoebae, ciliates, and flagellates) that show dramatic variation in DNA content throughout their life cycles. Endoreplication of genomes enables cells to grow to large sizes and perhaps to also respond to changes in their environments. Few microeukaryotic life cycles have been studied in detail, which limits our understanding of how eukaryotes regulate and transmit their DNA across generations. Here, we use microscopy to study the life cycle of Allogromia laticollaris strain CSH, an early-diverging lineage within the Foraminifera (an ancient clade of predominantly marine amoebae). We show that DNA content changes significantly throughout their life cycle and further describe an unusual process called Zerfall, by which this species reorganizes a large nucleus with up to 12,000 genome copies into hundreds of small gametic nuclei, each with a single haploid genome. Our results are consistent with the idea that all eukaryotes demarcate germline DNA to pass on to offspring amidst more flexible somatic DNA and extend the known diversity of eukaryotic life cycles.

Continuous reproduction of planktonic foraminifera in laboratory culture.

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.

Assigning the unassigned: A signature-based classification of rDNA metabarcodes reveals new deep-sea diversity.

Environmental DNA metabarcoding reveals a vast genetic diversity of marine eukaryotes. Yet, most of the metabarcoding data remain unassigned due to the paucity of reference databases. This is particularly true for the deep-sea meiofauna and eukaryotic microbiota, whose hidden diversity is largely unexplored. Here, we tackle this issue by using unique DNA signatures to classify unknown metabarcodes assigned to deep-sea foraminifera. We analyzed metabarcoding data obtained from 311 deep-sea sediment samples collected in the Clarion-Clipperton Fracture Zone, an area of potential polymetallic nodule exploitation in the Eastern Pacific Ocean. Using the signatures designed in the 37F hypervariable region of the 18S rRNA gene, we were able to classify 802 unassigned metabarcodes into 61 novel lineages, which have been placed in 27 phylogenetic clades. The comparison of new lineages with other foraminiferal datasets shows that most novel lineages are widely distributed in the deep sea. Five lineages are also present in the shallow-water datasets; however, phylogenetic analysis of these lineages separates deep-sea and shallow-water metabarcodes except in one case. While the signature-based classification does not solve the problem of gaps in reference databases, this taxonomy-free approach provides insight into the distribution and ecology of deep-sea species represented by unassigned metabarcodes, which could be useful in future applications of metabarcoding for environmental monitoring.

Assessment of the ecological quality status of the Sepetiba Bay (SE Brazil): When metabarcoding meets morphology on foraminifera.

In recent years, the region surrounding Sepetiba Bay (SB; SE Brazil) has become a hub of intense urban expansion and economic exploitation in response to ore transport and industrial and port activities. As a result, contaminants have been introduced into the bay, leading to an overall worsening of the environmental quality. The present work applies for the first time a foraminiferal morphology-based approach (M) and eDNA-based metabarcoding sequencing (G), along with geochemical data to assess the ecological quality status (EcoQS) in the SB. Principal component analysis shows that the eDNA and morphospecies diversity as well as most of the taxa relative abundance decline in response to the environmental stress (ES) gradient related to total organic carbon (TOC) and metal pollution. Based on ecological indices, Exp(H'bc) (G), Exp(H'bc) (M), foraminifera ATZI marine biotic index (Foram-AMBI), Foram Stress Index (FSI), and geochemical indices (TOC and Potential Ecological Risk Index), the lowest values of EcoQS (i.e., bad to moderate) are inferred in the innermost part of the SB. Despite minor discrepancies among the six EcoQS indices, an agreement has been found for 63% of the stations. To improve the agreement between the ecological indices, it is necessary to fill the gap in species ecology; information on the ecology of many species is still unknown. This work reinforces the importance of molecular analysis and morphological methods in environmental impact studies and confirms the reliability of foraminiferal metabarcoding in EcoQS assessment. This is the first study evaluating the EcoQS in the South Atlantic by using combined foraminiferal eDNA metabarcoding with morphological data.