A deep-learning automated image recognition method for measuring pore patterns in closely related bolivinids and calibration for quantitative nitrate paleo-reconstructions.

Eutrophication is accelerating the recent expansion of oxygen-depleted coastal marine environments. Several bolivinid foraminifera are abundant in these oxygen-depleted settings, and take up nitrate through the pores in their shells for denitrification. This makes their pore density a possible nitrate proxy. This study documents three aspects related to the porosity of bolivinids. 1. A new automated image analysis technique to determine the number of pores in bolivinids is tested. 2. The pore patterns of Bolivina spissa from five different ocean settings are analysed. The relationship between porosity, pore density and mean pore size significantly differs between the studied locations. Their porosity is mainly controlled by the size of the pores at the Gulf of Guayaquil (Peru), but by the number of pores at other studied locations. This might be related to the presence of a different cryptic Bolivina species in the Gulf of Guayaquil. 3. The pore densities of closely related bolivinids in core-top samples are calibrated as a bottom-water nitrate proxy. Bolivina spissa and Bolivina subadvena showed the same correlation between pore density and bottom-water nitrate concentrations, while the pore density of Bolivina argentea and Bolivina subadvena accumeata is much higher.

16S rRNA Gene Metabarcoding Indicates Species-Characteristic Microbiomes in Deep-Sea Benthic Foraminifera.

Foraminifera are unicellular eukaryotes that are an integral part of benthic fauna in many marine ecosystems, including the deep sea, with direct impacts on benthic biogeochemical cycles. In these systems, different foraminiferal species are known to have a distinct vertical distribution, i.e., microhabitat preference, which is tightly linked to the physico-chemical zonation of the sediment. Hence, foraminifera are well-adapted to thrive in various conditions, even under anoxia. However, despite the ecological and biogeochemical significance of foraminifera, their ecology remains poorly understood. This is especially true in terms of the composition and diversity of their microbiome, although foraminifera are known to harbor diverse endobionts, which may have a significant meaning to each species' survival strategy. In this study, we used 16S rRNA gene metabarcoding to investigate the microbiomes of five different deep-sea benthic foraminiferal species representing differing microhabitat preferences. The microbiomes of these species were compared intra- and inter-specifically, as well as with the surrounding sediment bacterial community. Our analysis indicated that each species was characterized with a distinct, statistically different microbiome that also differed from the surrounding sediment community in terms of diversity and dominant bacterial groups. We were also able to distinguish specific bacterial groups that seemed to be strongly associated with particular foraminiferal species, such as the family Marinilabiliaceae for Chilostomella ovoidea and the family Hyphomicrobiaceae for Bulimina subornata and Bulimina striata. The presence of bacterial groups that are tightly associated to a certain foraminiferal species implies that there may exist unique, potentially symbiotic relationships between foraminifera and bacteria that have been previously overlooked. Furthermore, the foraminifera contained chloroplast reads originating from different sources, likely reflecting trophic preferences and ecological characteristics of the different species. This study demonstrates the potential of 16S rRNA gene metabarcoding in resolving the microbiome composition and diversity of eukaryotic unicellular organisms, providing unique in situ insights into enigmatic deep-sea ecosystems.

Metabarcoding Insights Into the Trophic Behavior and Identity of Intertidal Benthic Foraminifera.

Foraminifera are ubiquitous marine protists with an important role in the benthic carbon cycle. However, morphological observations often fail to resolve their exact taxonomic placement and there is a lack of field studies on their particular trophic preferences. Here, we propose the application of metabarcoding as a tool for the elucidation of the in situ feeding behavior of benthic foraminifera, while also allowing the correct taxonomic assignment of the feeder, using the V9 region of the 18S (small subunit; SSU) rRNA gene. Living foraminiferal specimens were collected from two intertidal mudflats of the Wadden Sea and DNA was extracted from foraminiferal individuals and from the surrounding sediments. Molecular analysis allowed us to confirm that our foraminiferal specimens belong to three genetic types: Ammonia sp. T6, Elphidium sp. S5 and Haynesina sp. S16. Foraminiferal intracellular eukaryote communities reflected to an extent those of the surrounding sediments but at different relative abundances. Unlike sediment eukaryote communities, which were largely determined by the sampling site, foraminiferal intracellular eukaryote communities were driven by foraminiferal species, followed by sediment depth. Our data suggests that Ammonia sp. T6 can predate on metazoan classes, whereas Elphidium sp. S5 and Haynesina sp. S16 are more likely to ingest diatoms. These observations, alongside the use of metabarcoding in similar ecological studies, significantly contribute to our overall understanding of the ecological roles of these protists in intertidal benthic environments and their position and function in the benthic food webs.