Schopf et al.—Berkuta and Chulaktau microbiotas
(Nikon,Melville, NY) and an Olympus DP12 Microscope Digital Camera (Olympus, Melville, NY). At GIN, transmitted-light optical photomicrographs were acquired by use of an RME 5 microscope (Mikroskop Technik, Rathenow, Germany) equipped with a Cannon EOS 300D digital camera (Canon, Tokyo, Japan) and a ZeissAxio ImagerA1microscope (#3517002390) equipped with an AxioCam MRc 5 digital camera (Carl Zeiss, Jena, Germany).
Confocal laser scanning microscopy.—CLSM images were obtained with an Olympus Fluoview 300 confocal laser scanning biological microscope system equipped with two Melles Griot lasers, a 488 nm, 20mWoutput argon ion laser and a 633 nm, 10mW output helium-neon laser (Melles Griot, Carlsbad, CA). The images were acquired using a 60×
oil-immersion objective (numerical aperture 1.4) and fluorescence-free microscopy immersion oil (Cargille Labora- tories, Cedar Grove, NJ) with the use of filters in the light-path, to remove wavelengths <510 nm (for 488nm laser excitation) and <660nm (for 633nm laser excitation) from the laser- induced fluorescence emitted by the specimen, and of the Olympus Protocol Processor, to maximize useful data through- out the specimen. To provide maximum spatial information, most images were deconvoluted by use of the computer program Huygens Essential v3.2 (Scientific Volume Imaging, the Netherlands) and were subsequently processed by use of the VolView v2.0 three-dimensional-rendering computer program (Kitware, Clifton Park, NY) that permits their vertical and horizontal manipulation.
Raman and fluorescence spectroscopy.—Analyses of the fossils and associated minerals were carried out at UCLA by use of a T64000 (JY Horiba, Edison, NJ) triple-stage laser-Raman
system that has macro-Raman and confocal micro-Raman and fluorescence spectroscopic capabilities. This system permitted acquisition of point spectra and of Raman and fluorescence images that display the two-dimensional spatial distribution of molecular-structural components of the specimens and their associated matrix, with the varying intensities in such images corresponding to the relative concentrations of the molecular structures detected. Due to the confocal capability of this system, use of a 50× objective (having an extended working distance of 10.6mm and a numerical aperture of 0.5) provided a horizontal resolution of ~1.5 µm and a vertical resolution of 2–3 µm, with use of a 100× objective (working distance: 3.4mm; numerical aperture: 0.8) providing a horizontal resolution of <1 µm and a vertical resolution of ~1 µm. For thin sections overlain by a glass cover slip, a 40× objective having a cover slip correction-collar was used (working distance: 4.2mm; numerical aperture: 0.6) that provided horizontal and vertical resolution similar to that noted above. A Coherent Innova (Santa Clara, CA) argon ion laser provided excitation at 457.9nm permitting Raman data to be obtained over a range from ~300 to ~3000 cm−1 by use of a single spectral window centered at 1800cm−1. Fluorescence spectra were acquired over the wavelength range extending from <465 to ~900 nm. For Raman and fluorescence imaging, specimen-
containing thin sections lacking an overlying cover slip were veneered by a thin layer of the fluorescence-free microscopy
705 immersion oil noted above, the presence of which has been
shown to have no discernable effect on the Raman and fluorescence spectra acquired (Schopf and Kudryavtsev, 2010; Schopf et al., 2005), and the fossil was centered in the path of the laser beam projected through the microscope of the system. The laser power used for Raman imaging was ~1–8mWover an ~1 µm spot, an instrumental configuration well below the threshold resulting in radiation damage to such specimens
(Schopf et al., 2005). Two-dimensional spectroscopic fluorescence images that show the spatial distribution of fossil- permineralizing and -infilling apatite, rendered fluorescent by the presence of samarium+3 replacing both its calcium I and Ca II sites, were acquired in a narrow, ~20-nm broad spectral window centered at ~603nm to include both the ~597nm and 605nm bands. For euhedral crystals of fossil-encrusting apatite that exhibit spatially distinct regions of Sm+3-substituted Ca I and Ca II sites, images were acquired in 4- to 6-nm broad spectral windows centered at ~597 nm, for Ca I-replaced sites, and at ~605 nm, for Ca II-replaced sites.
Measurement of specimens and notations used in taxonomic descriptions.—At GIN, the specimen and cell sizes reported here were measured by use of Zeiss Axio Imager A1 software (Carl Zeiss, Jena, Germany). Where appropriate, the taxonomic descriptions indicate the mean cell size of the population measured (μ), standard deviation of the population(σ), the relative standard deviation of the population (RSD, where RSD = [σ/μ] × [100%]), and the number of measured specimens (n). For many of the spheroidal morphotypes, the taxonomic description indicates the divisional dispersion index (DDI), a metric designed to interrelate the endpoints of a population size range defined as “the least number of sequential vegetative divisions required to mathematically ‘reduce’ the largest cell of a population to the smallest cell of that popula- tion” and a genetically determined trait shown for 473 species and varieties of modern coccoidal prokaryotes and eukaryotes to cluster in the range from 2 to 4 with the great majority (~94%) having DDIs of 6 or less (Schopf 1976; 1992a, p. 1159).
Terminology.—We here use the term ‘cell’ to refer to spheroidal or ellipsoidal bodies defined by distinct carbonaceous walls that we interpret to be the originally cytoplasm-containing vegetative units of unicellular or colonial chroococcacean cyanobacteria and/or eukaryotic microalgae, or the similarly distinct spheroidal to box-like segments that comprise the trichomes of filamentous cyanobacteria. In general, this termi- nology is the same as that in our earlier papers on the Tamda Group-underlying Chichkan microbiota (Schopf et al., 2010a; Sergeev and Schopf, 2010).
Biological composition of the Berkuta and Chulaktau microbiotas
The taxonomic composition of the Berkuta and Chulaktau microfossil assemblages is summarized in Fig. 6. The 27 distinct entities recognized (illustrated in Figs. 3–14) are grouped into four morphological categories: (1) mat-forming filamentous cyanobacteria, (2) colonial and single-celled chroococcacean
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