Global radiation in a rare biosphere soil diatom

Soil micro-organisms drive the global carbon and nutrient cycles that underlie essential ecosystem functions. Yet, we are only beginning to grasp the drivers of terrestrial microbial diversity and biogeography, which presents a substantial barrier to understanding community dynamics and ecosystem functioning. This is especially true for soil protists, which despite their functional significance have received comparatively less interest than their bacterial counterparts. Here, we investigate the diversification of Pinnularia borealis, a rare biosphere soil diatom species complex, using a global sampling of >800 strains. We document unprecedented high levels of species-diversity, reflecting a global radiation since the Eocene/Oligocene global cooling. Our analyses suggest diversification was largely driven by colonization of novel geographic areas and subsequent evolution in isolation. These results illuminate our understanding of how protist diversity, biogeographical patterns, and members of the rare biosphere are generated, and suggest allopatric speciation to be a powerful mechanism for diversification of micro-organisms.

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Policy information about availability of computer code Data collection Data analysis Eveline Pinseel, Wim Vyverman Apr 16, 2020 No software was used for data collection.

October 2018
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Ecological, evolutionary & environmental sciences study design
All studies must disclose on these points even when the disclosure is negative.
Study description -BAMM v2.5.0 was used to run the BAMM program (available at http://bamm-project.org/) -Mesquite v3.61 was used to perform maximum likelihood ancestral state reconstructions of habitat type (available at http:// www.mesquiteproject.org/). This study consists of a global sampling of environmental samples containing the diatom species complex P. borealis. From these samples, diatom cultures were established. These were sequenced for various genes using Sanger sequencing. The sequences were subsequently used to build phylogenetic trees, assess diversity levels of P. borealis, and to investigate the diversification and biogeographic history of P. borealis. In addition, part of the samples was used for environmental DNA analysis using metabarcoding. Together with the already existing dataset, the newly developed dataset included 867 monoclonal cultures of the P. borealis species complex from various locations worldwide, as well as 132 environmental samples which included P. borealis cells.
In this study, we were interested in the diversity and evolutionary history of the diatom species complex P. borealis. Therefore, our sample design was designed to retrieve a maximum number of different species belonging to the complex, and to simultaneously recover intraspecific diversity to aid with the automated molecular species delimitation methods. Our sampling thus represents a representative subset of the P. borealis species present on a global scale.
As we aimed to provide an inventory of the global diversity of the P. borealis species complex, our sampling strategy included a) isolating P. borealis from soils sampled at the global scale to at least retrieve an equal number of varieties and formae described within the P. borealis complex by means of morphological features (see Kociolek JP, et al. 2018. DiatomBase. Accessed at http:// www.diatombase.org/ on 2018-02-28; 66 varieties and formae of P. borealis), and b) monitoring the accumulation of new molecular lineages as a function of investigated soil samples/strains. Standardized intensive screening (see 'Data collection') of soil samples ensured that presence/absence of borealis could be determined with confidence. However, the number of molecular lineages accumulated as a function of the number of investigated soil samples did not level off, despite analysis of over 1,500 soil samples. In this paper we do not claim to provide a complete view of P. borealis global diversity as the results of rarefaction analysis suggest the existence of numerous additional species, provided additional sampling. In fact, this unexpected finding is one of the novelties which makes our paper a game changing publication.
More in detail, for each sampling locality, multiple samples were collected randomly for establishing monoclonal cultures. Care was taken to include the different microhabitats that were present in the area. A various number of samples was collected per area, depending on time-availability, and the number of different microhabitats that were present. Samples for diatom cultures were collected from terrestrial mosses (above-and belowground parts), soils (top layer, ± upper 2 cm), and littoral sediments from lakes and ponds. All samples were stored in sterile falcon tubes (15 mL/50 mL) or sterile sampling bags. All samples were stored dark, and if possible, cool (< 10°C) during transport. No chemicals were added. Upon arrival in the lab, all samples were stored at 4°C, with exception of samples from subtropical regions (which were stored at 18°C).
For several of the samples collected for culture establishment of P. borealis, duplicate samples were collected in the field for environmental DNA analysis. Samples for environmental DNA analysis were collected from terrestrial mosses (above-and belowground parts), soils (top layer, ± upper 2 cm), and littoral sediments from lakes and ponds, as described above. All samples were stored dark, and if possible, frozen (-20°C). When samples could not be frozen, Sucrose Lysis Buffer (SLB; 20 mM EDTA, 200 mM NaCl, 0.75M sucrose, and 50 mM Tris-HCl at pH 9) was added to prevent biological activity, and to preserve DNA quality. Upon arrival in the lab, all samples were frozen at -80°C, prior to DNA extraction. Small quantities of the natural material (subsamples) were incubated for several weeks to months in WC medium, without pH adjustment or vitamin addition, at 4°C (for polar and temperate regions) or 18°C (for subtropical regions), 5 -10 µmol photons m-2 s-1 and a 12:12h (light:dark) cycle. Although the abundances of P. borealis cells were low in the overall majority of the samples, this was accommodated by careful sample treatment by the first author (E.P.). All environmental samples were subsampled in multiple wells of 12-well plates. In doing so, care was taken to take material from different parts of the sample, and if the sample was heterogeneous (for example, a mix of soil and moss), multiple subsamples from these different parts were taken. These samples were subsequently screened repeatedly in a light microscope over a course of several weeks. In case only dead valves of P. borealis were observed, samples were screened over longer time periods (up to four to six months). Although time-consuming, this approach ensured that the chances of observing living P. borealis cells were maximized.
Isolations of P. borealis cells were performed whenever it became possible to find living cells. Monoclonal cultures were established by isolating single cells under an Olympus SZX9 stereomicroscope using a needle and a micropipette. Cultures were grown in WC medium at standard culture conditions of 18°C, 5-10 µmol photons m-2 s-1 and a 12:12h (light:dark) cycle, and reinoculated when reaching late exponential phase. When sufficient biomass was obtained, subsamples for morphological and molecular analysis were taken.
All culture work was performed by E.P., under initial guidance of P.V. Molecular data analysis was performed by E.P. and several lab technicians (listed in acknowledgment).
The spatial scale of this study is global: samples were collected from all continents.
Samples were collected over a seven-year time period (between 2011 and 2017). The samples were collected during the growing season (spring -summer -autumn). The samples were collected once for each locality (no repeated sampling was done).
In this study, we aimed to provide a first overview of the global diversity of the diatom species complex surrounding P. borealis. To this end, the geographic coverage had to be maximized, and as a consequence repeated sampling was not feasible. Sampling was