This work explores the potential of multi-color Photoactivated Localization Microscopy (PALM) imaging to probe sub-diffraction limit interactions between proteins with spectrally separated labels. Using a PALM setup built around a commercial microscope axially stabilized to nm-level, we determined the ultimate registration accuracy that could be achieved (10 nm) and compared the performance of three different pairs of fluorescent proteins that can be used in dual color PALM. Fusion constructs were cloned and imaged either in vitro or at the cell plasma membrane, allowing to identify a current limit to co-localization precision of approximately 30-40 nm. We identified the better performing pair and present a concluding perspective application to a co-clustering study.

Abstract. Neogloboquadrina pachyderma is the dominant planktonic foraminifera species in the polar regions. In the northern high-latitude ocean, it makes up more than 90 % of the total assemblages, making it the dominant pelagic calcifier and carrier of paleoceanographic proxies. To assess the reaction of this species to a future shaped by climate change and to be able to interpret the paleoecological signal contained in its shells, its depth habitat must be known. Previous work showed that N. pachyderma in the northern polar regions has a highly variable depth habitat, ranging from the surface mixed layer to several hundreds of metres below the surface, and the origin of this variability remained unclear. In order to investigate the factors controlling the depth habitat of N. pachyderma, we compiled new and existing population density profiles from 104 stratified plankton tow hauls collected in the Arctic and the North Atlantic oceans during 14 oceanographic expeditions. For each vertical profile, the depth habitat (DH) was calculated as the abundance-weighted mean depth of occurrence. We then tested to what degree environmental factors (mixed-layer depth, sea surface temperature, sea surface salinity, chlorophyll a concentration, and sea ice concentration) and ecological factors (synchronized reproduction and daily vertical migration) can predict the observed DH variability and compared the observed DH behaviour with simulations by a numerical model predicting planktonic foraminifera distribution. Our data show that the DH of N. pachyderma varies between 25 and 280 m (average ∼100 m). In contrast with the model simulations, which indicate that DH is associated with the depth of chlorophyll maximum, our analysis indicates that the presence of sea ice together with the concentration of chlorophyll a at the surface have the strongest influence on the vertical habitat of this species. N. pachyderma occurs deeper when sea ice and chlorophyll concentrations are low, suggesting a time-transgressive response to the evolution of (near) surface conditions during the annual cycle. Since only surface parameters appear to affect the vertical habitat of N. pachyderma, light or light-dependant processes might influence the ecology of this species. Our results can be used to improve predictions of the response of the species to climate change and thus to refine paleoclimatic reconstructions.

The planktonic foraminifera genus Globigerinoides provides a prime example of a species-rich genus in which genetic and morphological divergence are uncorrelated. To shed light on the evolutionary processes that lead to the present-day diversity of Globigerinoides, we investigated the genetic, ecological and morphological divergence of its constituent species. We assembled a global collection of single-cell barcode sequences and show that the genus consists of eight distinct genetic types organized in five extant morphospecies. Based on morphological evidence, we reassign the species Globoturborotalita tenella to Globigerinoides and amend Globigerinoides ruber by formally proposing two new subspecies, G. ruber albus n.subsp. and G. ruber ruber in order to express their subspecies level distinction and to replace the informal G. ruber “white” and G. ruber “pink”, respectively. The genetic types within G. ruber and Globigerinoides elongatus show a combination of endemism and coexistence, with little evidence for ecological differentiation. CT-scanning and ontogeny analysis reveal that the diagnostic differences in adult morphologies could be explained by alterations of the ontogenetic trajectories towards final (reproductive) size. This indicates that heterochrony may have caused the observed decoupling between genetic and morphological diversification within the genus. We find little evidence for environmental forcing of either the genetic or the morphological diversification, which allude to biotic interactions such as symbiosis, as the driver of speciation in Globigerinoides.

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