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Linking evolution and development: Synchrotron Radiation X-ray tomographic microscopy of
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planktic foraminifers
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DANIELA N. SCHMIDT1*, EMILY J. RAYFIELD1, ALEXANDRA COCKING1 and FEDERICA
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MARONE2
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School of Earth Sciences, University of Bristol, BS8 1RJ Bristol, UK; e-mails:
d.schmidt@bristol.ac.uk; e.rayfield@bristol.ac.uk; ac3059@bristol.ac.uk
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Swiss Light Source, Paul Scherrer Institut, 5232 Villingen PSI, Switzerland; email:
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federica.marone@psi.ch
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*
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Corresponding author
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Abstract: Making the link between evolutionary processes and development in extinct organisms is
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usually hampered by the lack of preservation of ontogenetic stages in the fossil record. Planktic
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foraminifers, which grow by adding chambers, are an ideal target organism for such studies since their
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test incorporates all prior developmental stages. Previously, studies of development in these
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organisms were limited by the small size of their early chambers. Here we describe the application of
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Synchrotron Radiation X-ray tomographic microscopy (SRXTM) to document the ontogenetic history
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of the foraminifers Globigerinoides sacculifer and Globorotalia menardii. Our SRXTM scans permit
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resolution at submicrometre scale, thereby displaying additional internal structures such as pores,
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dissolution patterns and complexity of the wall growth. Our methods provide a powerful tool to pick
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apart the developmental history of these microfossils and subsequently assist in inferring phylogenetic
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relationships and evolutionary processes.
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Key words: Synchrotron Radiation X-ray tomographic microscopy, planktic foraminifers, evolution,
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development
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SYNCHROTRON TOMOGRAPHY OF PLANKTIC FORAMINIFERS
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TRADITIONALLY, the fossilised adult of an organism is the target of palaeobiological studies of
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form, complexity, and morphological diversity (Carroll 2001). Over the last decades, studies have also
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begun to incorporate developmental stages (Arthur 2002) with the birth of evolutionary
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developmental biology, or Evo-Devo. The application of Evo-Devo to fossil material is often hindered
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by the incomplete preservation of successive ontogenetic stages of most organisms. Foraminifers, in
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contrast, hold exceptional potential for studies linking ontogeny with phylogeny as they grow by
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adding chambers (Rhumbler 1911), encompass all developmental stages into the adult form and can
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therefore provide an invaluable archive for developmental studies. Due to their excellent stratigraphic
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control, great abundance, global distribution and good fossilisation potential combined with a wealth
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of biological and environmental information, the fossil record of planktic foraminifers and their well
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understood phylogenies are an ideal archive of evolutionary experiments. Using foraminifers will
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therefore provide high quality data for studies which are currently based on assembled series of a
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number of specimens of species such as trilobites or dinosaurs, and address questions such as phases
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of ontogeny under selection or changes in timing of development (McKinney 1990, McKinney 1999).
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Unravelling the ontogenetic stages in foraminifera is time consuming or limited in its resolution using
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traditional methods. Early studies used projection x-ray microscopy to reveal internal morphology (Bé
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et al. 1969). While this method allowed observation of internal morphology without destruction of the
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specimen, it was limited in its applicability to the small early phases of development and thick walled
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species. Importantly, coarsely ornamented, heavily encrusted, or infilled tests cannot be analysed by
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this method and high-spired specimens often do not produce good enough images for analysis (Huber
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1994). A number of studies applied dissection of adult specimens using a micromanipulator, a slow
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and laborious process (Huang 1981; Sverdlove and Bé 1985; Huber 1994), while Brummer et al.
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(1986) defined ontogenetic stages based on an assembly of an ontogenetic series of specimens from
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plankton tows.
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In the last decade, theoretical ontogenetic growth patterns were derived from computer models of
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foraminiferal growth (e.g. Tyszka 2006), for example suggesting that early chambers in log-spirally
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coiled structures cannot follow a strict isometric volume growth pattern (Signes et al. 1993). These
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studies proposed that juvenile stages have to be more planispiral and contain more chambers per
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whorl than adult stages. To test these suggestions, high resolution images of all ontogenetic stages of
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one specimen are necessary to avoid individual growth differences.
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X-ray computed tomography (CT) provides the necessary resolution to allow for such studies by
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using multiple images from different orientations to assemblage a series of virtual slices through a
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specimen. For example, Speijer et al. (2008) used laboratory based X-ray computed tomography to
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unravel the ontogenetic history of the benthic foraminifer Pseudouvigerina sp. In X-ray tomographic
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microscopy, an X-ray beam is passed through the specimen several times from different angles and is
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differentially attenuated depending on the density of the sample material and its structural
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arrangement. A set of tomograms are computed from the attenuation images. This technique reveals
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internal morphological information of the study object in a non-destructive manner and without any
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specific sample preparation.
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In the investigation of foraminifera, the resolution achieved with CT imaging enables the
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identification and isolation of all individual chambers down to the first. In this way 3-D digital models
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of each life stage can be built and subsequently scrutinised for precise morphological analysis and
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measurement. The 3-D model presents a significant advantage compared to dissection methods as the
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models can be rotated to best expose chamber arrangement, position of the primary aperture,
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arrangements of pores and wall structures. This information is important for the understanding of
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phylogenetic relationships between foraminifers or changes in timing of development across
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evolutionary transitions. Additionally, this method allows linking evolutionary studies to
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environmental change, as morphometric and geochemical studies can be performed on the same
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specimen.
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In this study we have employed synchrotron radiation X-ray tomographic microscopy (SRXTM) to
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image foraminifera rather than standard micro-CT methods. The high brilliance of synchrotron light
SYNCHROTRON TOMOGRAPHY OF PLANKTIC FORAMINIFERS
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provides increased spatial and temporal resolution compared to laboratory sources: detection of
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details as small as 1 micron in millimeter-sized samples is routinely possible within only few minutes.
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While this is also the case for high resolution micro-CT analysis, the monochromaticity of the used X-
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ray beam additionally enables precise attunement of beam energy to specimen properties and
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composition, making quantitative measurements of material properties possible and identification of
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different phases easier. Therefore, SRXTM allows beam hardening artefacts, distinctive for laboratory
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setups such as micro-CT with their lower flux and therefore broader energy spectrum, to be avoided.
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For a homogeneous cylindrical sample, beam hardening artefacts result in an artificial inhomogeneous
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grey level distribution with the centre darker than the borders, thereby hindering quantitative analysis.
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Using SRXTM, increased contrast and reduced noise are also promoted by the monochromatic beam
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and the high photon flux.
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An additional advantage of SRXTM over laboratory based X-ray computed tomography is the ability
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to use phase contrast for edge enhancement thanks to the coherence of synchrotron light. Edge
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enhancement offers improved accuracy in determining specimen boundaries and volumetric
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measurements as well as facilitating the visualization of internal structures in the foraminiferal wall
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such as the position of the organic layers, pores and dissolution features (Fig. 1).
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Taxonomy and stratigraphy of the investigated species
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We have applied SRXTM to two representatives of the major clades (Globigerinidae and
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Globorotaliidae) of extant planktic foraminifers, Globigerinoides sacculifer and Globorotalia
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menardii from Holocene sediment samples from the South Atlantic. Overall, foraminiferal
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morphology is rather conservative and a few basic variables suffice to describe most species (Berger
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1969). Each morphology is characteristic for a species and hence individual specimens can be used as
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representatives of the species. Measurements describing their form deviate by just a few percent
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within populations (Huber 1994).
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Brummer et al. (1987) noted that Gs. sacculifer showed the most pronounced morphological change
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of all investigated species and used it to define a five stage model of ontogeny from the proloculus,
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via the juvenile and neanic stages (acquisition of adult characters) to the adult stage (full expression of
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adult characters), and the terminal (remodelling of the surface structures). Recognition of these stages
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was based on sudden shifts in test size, apertural position, chamber shape and arrangement, and
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surface ornamentation (e.g., presence/absence of pore pits, pore distribution).
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Gs. sacculifer originated in the early Miocene from Gs. triloba via Gs. immaturus and Gs.
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quadrilobatus (Kennett and Srinivasan 1983). The group exhibits a cancellate surface structure
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(Kennett and Srinivasan 1983). Gs. sacculifer inhabits the mixed layer, though the deposition of the
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gametogenetic layer often happens near the thermocline (Bé 1980). The adult specimen has a low
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trochospire. Early chambers are small and sub-globular, whilst final and penultimate chambers are
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often elongated (Brady 1884). A sack-shaped final chamber is often formed but culturing experiments
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have shown that the ‘trilobus’ (without the sack-shaped final chamber) and ‘sacculifer’ morphotype
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are the same biological species (Hemleben et al. 1989). The adult surface is covered with regular
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subhexagonal pore pits. The primary aperture is interio-marginal and umbilical and possesses a
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distinct arch bordered by a rim. The spiral side shows prominent supplementary apertures (Kennett
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and Srinivasan 1983). The maximum size of the species can exceed 1100 µm (Schmidt et al. 2004).
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Gs. sacculifer’s transition to Orbulina universa is a classical example of sympatric divergence
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(Pearson et al. 1997).
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Gr. menardii belongs to the macro-perforate non-spinose species which descended from
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Gr. praescitula, via Gr. (M.) archeomenardii and Gr. (M.) premenardii in the mid-Miocene (Kennett
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and Srinivasan 1983). It is the largest living planktic foraminiferal species with sizes up to 1500 µm
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(Schmidt, et al. 2004). The adult is low trochspiral, compressed with a lobulate periphery and strongly
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curved sutures. The axial periphery is acute with a prominent keel. The surface is densely perforated
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with circular pores. It can facultatively harbour symbionts and lives in the deeper part of the mixed
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layer (Hemleben, et al. 1989). During its development the shape becomes more compressed
SYNCHROTRON TOMOGRAPHY OF PLANKTIC FORAMINIFERS
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(Schweitzer and Lohmann 1991) which has been related to its depth migration in the water column
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(Fok-Pun and Komar 1983).
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METHODS
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A well-preserved representative adult specimen from each species was chosen for Synchrotron
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Radiation X-ray tomographic microscopy (SRXTM). Each specimen was mounted upon a 3 mm brass
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stub using a small amount of dilute PVA glue. SRXTM was performed at the TOMCAT beamline
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(Stampanoni et al. 2006) at the Swiss Light Source (SLS) at the Paul Scherrer Institut in Villigen,
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Switzerland. For these experiments, the X-ray beam energy was set to 17.5 keV to optimise for
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maximum contrast. The microscope magnification was set to 10x and the data was binned twice, a
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process which combines adjacent pixels (at two times binning combining 4 adjacent pixels to a single
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pixel). This binning procedure decreases the spatial resolution of the image, but improves the signal-
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to-noise ratio, whilst decreasing data acquisition and post-processing time. The resulting voxel size
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for both species datasets was 1.4 μm. We therefore estimate the error in our measurements of the
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chamber size to be in the order of two voxels, i.e. ~3 µm.
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For each dataset 721 projections equi-angularly spaced over 180° were acquired. Tomographic
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reconstructions were computed on-site using a highly optimized routine based on the Fourier
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Transform method (Marone et al. 2010). Each final tomographic volume consisted of a series of TIFF
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images, representing 200 (Gt. menardii) and 427 (Gs. sacculifer) sequential axial slices through the
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specimens.
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The reconstructed tomographic volume was imported into the 3D visualisation software Amira 4.0
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(Mercury Computer Systems, www.tgs.com, Fig. 2). Different grey levels are assigned to each pixel
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as a measure of the degree of interaction of the different sample components with the monochromatic
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X-ray beam. As such, the test was digitally isolated from any residual sediment with differential
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attenuation properties. The homogenous nature of the calcite test resulted in similar X-ray attenuation
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properties through the specimen, therefore successive chambers were manually isolated and separated.
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Each ontogenetic stage was labelled as a distinct entity (Fig. 2). While current levels of resolution are
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able to visualise and distinguish the internal organic layers within the test, separating each of them
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individually would have been extremely time consuming. Three-dimensional surface renderings of
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each developmental stage were created for measurement and visualisation of the ontogenetic
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sequence.
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In order to envisage ontogenetic shape change, a series of 2D TIFF images were obtained from the
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Amira reconstructions in spiral, umbilical and side views for (i) each ontogenetic stage for both
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species, and (ii) each isolated chamber for both species. In order to document proportional change
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during growth and chamber acquisition, length and width measurements in spiral view were taken of
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the whole organism at each ontogenetic stage, and for each chamber, using ImageProPlus
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(MediaCybernetics).
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RESULTS
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Gs. sacculifer
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The imaged specimen of Gs. sacculifer has a maximum height of 705 μm (Fig. 3). The 1.4 μm scan
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resolution leads to the pixellated appearance of first chambers. The proloculus, the first chamber, is
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nearly spherical at 18 by 16 μm, thereby strongly contradicting earlier work by Banner and Blow
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(1960) who suggested a proloculus size of 30 µm but within the range of values of Parker (1962) with
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a range of 10 to 16 µm. It is likely that the dissection by Banner and Blow did not expose the
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proloculus but a later chamber (based on our measurements somewhere between chambers 5 to 8),
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while the process of changing the calcium carbonate tests to calcium fluoride and mounting them in
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Canada balsam as performed by Parker is likely to lead to artefacts due to partial dissolution and
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projection, thereby highlighting the strength of our method. The deuteroconch, the second chamber, is
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smaller than the proloculus as previously described by Huang (1981), Sverdlove & Bé (1985) and
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Huber (1994). The third and fourth chambers are similar in size with 18 µm. The 5th chamber is
SYNCHROTRON TOMOGRAPHY OF PLANKTIC FORAMINIFERS
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significantly larger and shows a change from roundish to more triangular shape. The chambers
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subsequently become increasingly inflated. The coiling increases in its trochospiral nature (involute
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on the spiral side) from the 8th stage onwards leading to an overall globorotaliid shape, confirming
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the proposition by Signes et al. (1993) based on computer models that juvenile stages have to be more
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planispiral and contain more chambers per whorl than adult stages. Specifically, starting with the 8th
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chamber the juvenile has the typical seven chambers per whorl (Brummer, et al. 1987) and hence
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significantly more than the adult and terminal phase (Fig. 3) confirming Parker’s (1962) suggestion of
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the comparably high number of juveniles chamber per whorl for this species.
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Chamber 12 displays a strong difference in surface texture changing from smooth to the development
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of the cancellate surface typical for the lineage. This morphological change indicates the first steps in
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the transition from the juvenile to the neanic stage associated with a slight increase in the chamber
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extension rate (Fig. 4A, arrow). The new chamber is strongly pitted, indicating the development of
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large pores. The shape of the newly added chamber is more roundish thereby starting to resemble to
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globigerinid shape. The 14th chamber is the first to demonstrate the typical round chambers of the
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adult specimen. The addition of a second round chamber at stage 15 leads to an overall shape
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resembling the adult morphology; thereby finalising the transition from neanic to adult. At the same
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time the primary aperture moves from the extra-umbilical to the umbilical position. This transition is
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associated with a strong increase in chamber and test size (Fig. 4A, arrow). At stage 16 the first
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secondary apertures (the characteristics for the genus and the adult stage) are clearly visible. The
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primary aperture / secondary apertures are significantly smaller at this stage than in the terminal one.
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The aperture in chamber 17 displays the typical distinct arch and only on the final chamber is the
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aperture bordered by a rim.
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The size of Gs. sacculifer is dominated by the last four chambers, the addition of which leads to a
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growth from 150 µm to more than 700 µm (Figs 3 and 4). While chambers 12 to 14 are already larger
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than their predecessors, their size increase is significantly less than the later, last chambers (Figs 3 and
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4).
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Gr. menardii
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In contrast to Gs. sacculifer, the overall shape of Gr. menardii is stable throughout ontogeny (Fig. 6).
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The proloculus appears to be ovoid and the deuteroconch is separated from the proloculus by a flat
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wall. The proloculus shape of Gr. menardii is likely an artefact of the limits of scan resolution and
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reconstruction, although Hemleben et al. (1977) point out that the proloculus retains a highly flexible
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wall due to minimal calcification, which would allow it to deform during chamber addition. The
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proloculus size in Gr. menardii is larger than in Gs. sacculifer by ~20% and likely the cause for the
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larger final size despite a similar chamber number as suggested in earlier work that the size of the
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proloculus strongly influences the final size and chamber arrangement of the test (Sverdlove and Be
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1985, Huber 1994). The first four chambers are very similar in size (Figs 4B and 5). From chamber
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five onwards there is a slight increase in growth relative to the earlier chambers. The overall shape is
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significantly more inflated on the umbilical side. The periphery is clearly lobate. The axial periphery
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becomes increasingly acute from the 6th chamber onwards. From chamber eight onwards, the final
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whorl contains six chambers. This persists until the adult stage of chamber 15, when the final whorl is
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reduced to five chambers. The increase in chamber expansion rate starting with stage 9 (Fig. 4B
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arrow) is not related to an overall change in shape. From the 10th chamber onwards, the overall shape
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becomes progressively compressed, a process which is completed in the penultimate chamber. The
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spiral side starts to curve slightly starting with the 11th chamber. From the 12th chamber onwards, the
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keel has the typical thick appearance of the adult specimen, the sutures on the spiral sides are raised,
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the wide umbilicus starts to develop and a lip is clearly visible finalising the transition to the adult
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specimen. The 13th and all subsequent chambers are significantly larger than the previous ones (Fig.
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4B arrow). Chamber 15 shows the change from a large round to a low arched aperture.
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In Gr. menardii overall size rapidly increases with the addition of chambers 15 and 16. The
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kummerform growth of the Gr. menardii specimen is highlighted by the smaller width and length of
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the final, 17th chamber (Figs 2 and 3) compared to the penultimate (16th) chamber.
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SYNCHROTRON TOMOGRAPHY OF PLANKTIC FORAMINIFERS
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DISCUSSION
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The reconstruction of the ontogeny of the entire specimen, in contrast to earlier work which
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assembled a number of specimens of different ontogenetic stages, allows for precise single-specimen
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measurements of growth during ontogeny, and thereby a precise comparison of growth patterns in a
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range of foraminiferal species from the first chamber onwards.
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Both species show an exponential growth during their overall ontogeny (Fig. 4) though for the first
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stages (9 stages in Gs. sacculifer and 8 in Gr. menardii) the growth is not distinguishable from linear.
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Overall, the growth trajectories in both species are astonishingly similar (Fig. 4). The overall test
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aspect ratio of both species shows less variability than the individual chamber form. The patterns in
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test to chamber aspect ratio can be loosely divided in three stages: (i) a rapid increase in test aspect
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ratio in the first 5 (Gr. menardii) or 6 chambers (Gs. sacculifer); (ii) the growth phase associated with
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the development of the acute shape keel in Gr. menardii, which displays the highest aspect ratio; (iii)
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the adult phase, which displays a reduction in aspect ratios again. The ratio of test size to chamber
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size for both species show strikingly similar patterns despite their difference in final morphology,
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taxonomic groups and ecology, highlighting the overall conservative nature of the foraminiferal
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morphology. This suggests strong underlying biological, structural or fluid mechanical constraints on
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their shape and changes to surface to volume ratios as suggested by Berger (1969). Berger stated that
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three basic variables suffice to build models of most foraminifers with the chamber ratio being the
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most important one emphasising the simplicity of the foraminiferal morphology. At a more detailed
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level differences do arise, such as the last chambers of Gs. sacculifer expanding much more rapidly
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(Fig. 4C), despite the fact that this specimen lacks the typical sacc-like final chamber.
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The general picture suggests near isometric growth, i.e. a linear relationship between length and
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width, when the overall growth trajectory of both specimens is considered, though both specimens
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analysed herein are wider than long (Fig. 4C, D). A more detailed plot of the aspect ratios for both
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species, though, shows variability (Fig. 6) for both chamber and test growth. This highlights no strict
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isometric growth. It confirms Signes et al. (1993) model that early chambers in log-spirally coiled
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structures cannot follow a strict isometric volume growth pattern. Their model suggests that juvenile
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stages are more planispiral and have more chambers per whorl than adult stages. Our results clearly
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confirm their model predictions for both number of chambers and changes in the overall
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trochospirality of the specimens. It is important to mention that, though variations in aspect ratio at
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the smaller growth stages are strongly influenced by the resolution and precision of the scans and may
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also be influenced by remodelling of the earlier chambers during growth, they are unlikely in the
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order of difference necessary to change the result of the analysis.
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The analysis of the juvenile stages in three dimensions allows us to assess in which way these
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resemble the phylogeny of the organism. The similarity of juvenile morphology between these two
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genetically and phylogenetically separated species has been described by Parker (1962), Huang
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(1981), and Brummer et al. (1987). Gr. (M.) menardii evolved from Gr. (M.) praemenardii which is
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smaller and with a less-district peripheral keel. The shape of the ancestor resembles stages 14 and 15
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of the ontogeny of Gr. (M.) menardii, suggesting a change in timing of development might have led to
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the transition from one species to the other. Continued development (McKinney 1986) may have been
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the mechanism leading to the evolution from Gr. (M.) praemenardii to Gr. (M.) menardii. Similarly,
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Stages 11 and 12 resemble the Gr. (M.) archeomenardii shape with a large apertural face, more
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roundish aperture and faint keel again suggesting that peramorphosis (i.e. continued growth) could
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have been the cause for the evolution of the lineage. Future work on the ontogeny of the earlier
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menardellids will help to determine if changes in the onset or end of development were involved in
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the evolution of the genus. In contrast, none of the earlier traits of Gs. triloba or Gs. immaturus are
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preserved in Gs. sacculifer. The change from the globorotaliid form to a globigerind form does not
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reflect the evolutionary lineage.
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The complexity of the early Gs. sacculifer stages poses an interesting question. Specialisation of a
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species is often inferred from the complexity of the shell with globigerinid ‘roundish’ species being
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the simple, generalist shape (Cifelli 1969; Norris 1991). This interpretation may be misleading
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considering the complexity of earlier developmental stages. If the ‘simple’ globigerinid shape
SYNCHROTRON TOMOGRAPHY OF PLANKTIC FORAMINIFERS
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envelopes an earlier more ‘evolved’ globorotaliid shape, then the link between morphology and
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complexity is a gross oversimplification. This begs the question whether the perceived expansion of
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morphological variance over time (Norris, 1991), which is solely defined by the adult specimen, will
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still persist when the entire morphological range during ontogeny is considered.
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Acknowledgements
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This work was made possible by a grant from the Paul Scherrer Institut Swiss Light Source via
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European Union FP6 to DNS and EJR (Proposal ID 20060780) and funding from the Royal Society
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via a University Research Fellowship to DNS. We would like to thank Andy Henderson for joining us
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in Switzerland, Neil Gostling for assistance with Amira and Aude Caromel for graphic assistance.
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SYNCHROTRON TOMOGRAPHY OF PLANKTIC FORAMINIFERS
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FIG. 1. Cross sections based on Synchrotron Radiation X-ray tomographic datasets. A, Orbulina
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universa (~410 µm). B, Neogloboquadrina pachyderma (~300 µm). C, Gloroboralia
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truncatulinioides (~640 µm). D, Neogloboquadrina dutertrei (~300 µm) displaying internal
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dissolution features in A and B, the geometry of pore spaces in C and the position of internal organic
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layers in C and D, highlighting the advantages of Synchrotron Radiation X-ray tomographic
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microscopy including phase contrast for the analysis of foraminifers.
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FIG. 2. Examples of a tomographic volume postprocessed in Amira 4.0, with each developmental
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chamber labelled with a different colour. A. surface reconstruction of Globorotalia menenardii, B.
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horizontal section and C. vertical section, Size of specimen ~1500 µm.
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FIG. 3. 3D rendering of Globigerinoides sacculifer. Proloculus (top left, scale bar represent 10 µm) to
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the terminal stage (bottom right, scale bar represents 156 µm). Each chamber is labelled in a distinct
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colour, each stage indicated by numbers below specimen. Pixel size = 1.4 microns in all stages.
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FIG. 4. Growth patterns of Globigerinoides sacculifer and Globorotalia menardii. Chamber number
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versus size [µm] for chamber width (grey circle), chamber length (open grey circle), test width
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(filled triangle) and test length (open triangle). A. Globigerinoides sacculifer, B, Globorotalia
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menardii. Size to width ratio. C, overall test size and D, chamber size for both species. C, D,
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Globigerinoides sacculifer open grey circles, Globorotalia menardii filled black circles. The 1:1
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relationship in C and D is indicated by the stippled line.
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FIG. 5. 3D rendering of Globorotalia menardii. Proloculus (top left, scale bar represents 10 µm) to
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the terminal stage (bottom right, scale bar represents 100 µm). Each chamber is labelled in a distinct
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colour, each stage indicated by numbers below specimen. Pixel size = 1.4 microns in all stages.
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FIG. 6. Aspect ratios of length to width for chambers (circles) and overall test (triangles). A ,
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Globigerinoides sacculifer. B, Globorotalia menardii . C, Aspect ratio for test versus chamber
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length (circle) and width (triangle) for Globigerinoides sacculifer (black) and Globorotalia menardii
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(grey).
17