Research Article: Advanced approach to analyzing calcareous protists for present and past pelagic ecology: Comprehensive analysis of 3D-morphology, stable isotopes, and genes of planktic foraminifers

Date Published: March 7, 2019

Publisher: Public Library of Science

Author(s): Yurika Ujiié, Katsunori Kimoto, Toyoho Ishimura, Lukas Jonkers.


Marine protists play an important role in oceanic ecosystems and biogeochemical cycles. However, the difficulties in culturing pelagic protists indicate that their ecology and behavior remain poorly understood; phylogeographic studies based on single-cell genetic analyses have often shown that they are highly divergent at the biological species level, with variable geographic distributions. This indicates that their ecology could be complex. On the other hand, the biomineral (calcareous) shells of planktic foraminifers are widely used in geochemical analyses to estimate marine paleoenvironmental characteristics (i.e., temperature), because the shell chemical composition reflects ambient seawater conditions. Among the pelagic protists, planktic foraminifers are ideal study candidates to develop a combined approach of genetic, morphological, and geochemical methods, thus reflecting environmental and ecological characteristics. The present study precisely tested whether the DNA extraction process physically and chemically affects the shells of the planktic foraminifer Globigerinoides ruber. We used a nondestructive method for analyzing physical changes (micro-focus X-ray computed tomography (MXCT) scanning) to compare specimens at the pre- and post-DNA extraction stages. Our results demonstrate that DNA extraction has no significant effect on shell density and thickness. We measured stable carbon and oxygen isotopes on the shell of each individual in a negative control or one of two DNA-extracted groups and detected no significant differences in isotopic values among the three groups. Moreover, we evaluated isotopic variations at the biological species level with regard to their ecological characteristics such as depth habitat, life stages, and symbionts. Thus, our examination of the physiochemical effects on biomineral shells through DNA extraction shows that morphological and isotopic analyses of foraminifers can be combined with genetic analysis. These analytical methods are applicable to other shell-forming protists and microorganisms. In this study, we developed a powerful analytical tool for use in ecological and environmental studies of modern and past oceans.

Partial Text

Marine protists are the most abundant eukaryotes in the pelagic realm; recent field-based studies have unveiled their high diversity and abundance in the photic and deep layers of the world’s oceans [1,2]. Several techniques, including in situ imaging and metagenomic analysis, have been used to assess the biomass and variability of marine protists along horizontal and vertical dimensions of the oceans [3,4]. The results of previous studies suggest that protists greatly affect marine ecosystems [1–4]. In particular, protists represent the main component of the marine food web and act as a driving force for biogeochemical cycles [5]; therefore, knowledge of their ecology and environmental responses is important to understanding these roles. Although transcriptome analyses have been conducted to examine gene expression in protists from an ecological point of view [6], such metadata-based approaches (e.g., transcriptome and metagenomic analyses) have been limited to culturable species. Since most protists are difficult to culture, their ecology has remained unknown to date.

In the present study, we made a thorough examination of the physiochemical effects of the processes used for DNA extraction in biomineral shells. Our objective was to establish a comprehensive method of analysis that integrates genetic, physical/morphological, and isotopic information for small, calcified microorganisms, such as planktic foraminifers. We developed a nondestructive analysis method, involving MXCT scanning, that was successful in obtaining accurate physical data (i.e., calcite density) and morphological images of the shells. Our results also showed that the DNA extraction process did not cause any physical changes in the shells. A microscale isotopic analytical system was used to measure the δ13C and δ18O values for single specimens. We designed a controlled experiment based on two groups, each of which experienced a different DNA extraction incubation time at 70°C, and a negative control group that did not undergo DNA extraction procedures. No significant differences in δ13C and δ18O values were observed among the three experimental groups, therefore demonstrating that the DNA extraction process did not cause isotopic changes in foraminiferal shells. The shell weight-δ13C regression slopes between the various genetic types of planktic foraminifers tend to differ from each other, and this is probably influenced by the ecological traits of biological species such as differences in the associated algal symbionts. These data could contribute to a better understanding of the vital effects on the stable isotopes of foraminifers and help us to more accurately reconstruct the environmental changes reflected in stable isotopes. Our study presents a powerful analytical tool for use with microscale samples. The physical/morphological and isotopic indicators complement the ecological aspects of unculturable pelagic protists and shell-forming microorganisms. These advances would support prospective studies of ecology and evolution in conditions of drastically changing pelagic environments.




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