The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing

Microbial ecology is plagued by problems of an abstract nature. Cell sizes are so small and population sizes so large that both are virtually incomprehensible. Niches are so far from our everyday experience as to make their very definition elusive. Organisms that may be abundant and critical to our survival are little understood, seldom described and/or cultured, and sometimes yet to be even seen. One way to confront these problems is to use data of an even more abstract nature: molecular sequence data. Massive environmental nucleic acid sequencing, such as metagenomics or metatranscriptomics, promises functional analysis of microbial communities as a whole, without prior knowledge of which organisms are in the environment or exactly how they are interacting. But sequence-based ecological studies nearly always use a comparative approach, and that requires relevant reference sequences, which are an extremely limited resource when it comes to microbial eukaryotes

Spatial structure in the “Plastisphere”: Molecular resources for imaging microscopic communities on plastic marine debris

Plastic marine debris (PMD) affects spatial scales of life from microbes to whales. However, understanding interactions between plastic and microbes in the "Plastisphere"-the thin layer of life on the surface of PMD-has been technology-limited. Research into microbe-microbe and microbe-substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI-FISH (combinatorial labelling and spectral imaging - fluorescence in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria, Alpha-, and Gammaproteobacteria) and four newly designed probes (targeting Bacteroidetes, Vibrionaceae, Rhodobacteraceae and Alteromonadaceae) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell-count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro-metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales

Phylogenetic diversity and evolutionary relatedness of alkenone-producing haptophyte algae in lakes: implications for continental paleotemperature reconstructions

Alkenones have been found in an increasing number of lakes around the world, making them a promising new tool for continental paleoclimate reconstruction. However, individual lakes may harbor different species of haptophyte algae with different sensitivities to temperature variations, thus presenting a significant challenge to the use of lacustrine alkenones for paleotemperature reconstructions. To explore the extent of lacustrine haptophyte diversity, we conducted the first comprehensive phylogenetic and geochemical survey of lacustrine alkenone producers. We sampled 15 alkenone-containing lake surface sediments from a variety of geographic locales and inferred identities of environmental sequences using 18S ribosomal RNA (rRNA) gene-based phylogenies. For two lakes, BrayaSø in southwest Greenland and Tso Ur on the Tibetan Plateau, we also analyzed both surface and downcore sediments to characterize haptophyte populations through time. In parallel with phylogenetic analyses, we determined the alkenone distributions (including C37/C38 ratios, and the presence/absence of C38 methyl ketones and C40 compounds) in all the samples. The resulting alkenone profiles from this study do not all align with traditional “marine” versus “coastal/lacustrine” alkenone profiles. Additionally, our genetic data indicate the presence of multiple haptophyte species from a single lake sediment sample; these distinct haptophyte populations could not be discerned from the alkenone profiles alone. These results show that alkenone profiles are not a reliable way to assess the haptophyte algae in lakes and that DNA fingerprinting is a preferred approach for species identification. Although closely related haptophyte species or subspecies may not warrant different temperature calibrations, our results emphasize the importance of genetic data for inferring haptophyte identities and eventually selecting alkenone–temperature calibrations for paleoclimate reconstructions

Successional blooms of alkenone-producing haptophytes in Lake George, North Dakota: Implications for continental paleoclimate reconstructions

Alkenone‐derived paleotemperature reconstruction holds great promise in lake environments. However, the occurrence of multiple species of alkenone‐producing haptophyte algae in a single lake can complicate the translation of alkenone unsaturation to temperature if each species requires an individual temperature calibration. Here, we present the first systematic monitoring of two alkenone‐producing haptophytes throughout the course of a seasonal cycle in Lake George, North Dakota, using a combined approach of DNA sequencing and alkenone lipid characterization. Field sampling revealed a nonoverlapping haptophyte succession, with both an early and late season haptophyte bloom event. Culturing experiments demonstrated that the two haptophyte species responsible for these blooms had statistically similar alkenone‐temperature responses, although the culture‐based calibrations were distinct from the in situ calibration. Bloom timing of each haptophyte species corresponded to surface‐water temperatures that differed by more than 10°C, revealing that changes in bloom intensities for each species will skew the sediment‐inferred temperatures to a different stage of the growth season. These results highlight the importance of accounting for bloom timing when interpreting alkenone‐derived temperatures in sediment cores, especially in lakes that experience large seasonal fluctuations in water column temperature and salinity