Biomineral electron backscatter diffraction for palaeontology

Electron backscatter diffraction (EBSD) originated in materials science and has transferred to biomineral research providing insight into fossil and modern biominerals. An electron microscopy technique, EBSD requires a fine polished sample surface where the electron beam diffracts in the first few lattice layers, identifying mineral, polymorph and crystallographic orientation. The technique is particularly well suited for the analysis of modern and fossil calcium carbonate biominerals, where it provides key insight into biological control of mineral formation such as in molluscs and brachiopods. EBSD readily identifies original and secondary mineralogy, which helps to inform our understanding of biomineral evolution such as the identification of original aragonite in Silurian trimerellid brachiopods. As a technique to identify and thus avoid the inclusion of secondary minerals in proxy organisms such as corals, EBSD can be used to ensure accuracy of palaeoproxy data. Even when fossil systems have no modern equivalents, EBSD can provide key data to determine functional mechanisms such as in the lenses of schizochroal eyes of phacopine trilobites. These few examples illustrate that EBSD is proving to be a valuable component of the palaeontology toolkit.     

Electron backscatter diffraction (EBSD) as a tool for detection of coral diagenesis

Fine-scale structures of intact modern and fossil coralline skeletons were analysed to determine alteration to secondary cements and phases using electron backscatter diffraction (EBSD). EBSD analysis revealed secondary aragonite cements in endolithic borings in the modern skeleton and whole dissepiments of the fossil skeleton replaced by calcite, despite X-ray diffraction (XRD) bulk analysis of the general area suggesting only aragonite was present. Non-destructive, in situ screening of coral samples by EBSD analysis provides a valuable tool for assessing the extent of alteration and can determine which areas may produce more reliable climate proxy data. Communicated by Geology Editor Dr. Bernhard Riegl     

The Application of Electron Backscatter Diffraction on Halide Perovskite Materials

Recently, the application of electron backscatter diffraction (EBSD) in halide perovskites has enabled the correlation of the micro‐structural arrangement of polycrystalline grains with other properties (optical, electrical, mechanical, and chemical) in a “pixel‐by‐pixel” approach. Most studies so far have used an ultra‐sensitive electron beam detector that has sensitivity thousands of times higher than a traditional scintillator screen and charge coupled device camera, enabling much lower beam currents. An alternative approach has been the use of low vacuum measurement conditions to avoid charge buildup that leads to damage. This review focuses on introducing the classical EBSD technique to the halide perovskite community, where it has been highly underutilized due to beaminduced damage in these relatively unstable materials. Recent research is used to dispel some common misconceptions about grain boundaries in halide perovskites and highlight what has been learned by comparing and correlating EBSD with other techniques. Additionally, the remaining limitations, development challenges, and future of the EBSD technique for halide perovskites are discussed. Successful utilization of the EBSD technique as a common characterization tool in the halide perovskite community will enable scientists and engineers to develop maps of cross correlated properties, helping to unlock the full potential of this complex material system.     

Bryozoans as taphonomic engineers,with examples from the Upper Ordovician (Katian) of Midwestern North America

A combination of encrusting calcitic bryozoans and early seafloor dissolution of aragonitic shells recorded in the Cincinnatian Series of the upper Midwest of North America allowed the preservation of abundant moulds of mollusc fossils bioimmured beneath the attachment surfaces of the bryozoans. We here call this preservational process ‘bryoimmuration’, defined as a bryozoan‐mediated subset of bioimmuration. The bryozoans moulded very fine details of the mollusc shells, usually with more accuracy than inorganic sediment moulds. Most of the bryozoans are heterotrypid trepostomes with robust low‐Mg calcite skeletons. The molluscs are primarily bivalves, gastropods, nautiloids and monoplacophorans with their originally aragonitic shells now dissolved. Many of the encrusting bryozoans are so thin and broad that they give the illusion of calcitic mollusc shells clinging to the moulds. Some molluscs in the Cincinnatian, especially monoplacophorans and epifaunal bivalves, would be poorly known if they had not been bryoimmured. Unlike internal and external moulds in sediment, bryoimmured fossils could be transported and thus record aragonitic faunas in taphonomic assemblages (e.g. storm beds) in which they would otherwise be rare or absent. In addition, bryoimmurations of aragonitic shells often reveal the ecological succession of encrustation on the shells by exposing the earliest encrusters and borings that were later overgrown. Bryoimmuration was common during the Late Ordovician because the calcite sea at the time quickly dissolved aragonitic shells on the seafloor before final burial, and large calcitic bryozoans very commonly used molluscs as substrates. Bryoimmuration is an important taphonomic process for preserving aragonitic faunas, and it reveals critical information about sclerobiont palaeoecology. Several Cincinnatian mollusc holotypes are bryoimmured specimens. Bryozoans involved in bryoimmuration enhance the preservation of aragonitic fauna and thus act as taphonomic engineers.     

Electron microscopy,electron diffraction,and element analysis of wet biological specimens

Temperature controlled differentially pumped environmental chambers now allow more routine examination of wet specimens in the electron microscope. A sensitive test of their efficiency is the ability to provide high resolution electron diffraction patterns from wet, unfixed protein microcrystals. Fortunately, wet specimens can be prepared with only a few tens of nanometers thickness of remaining water, so extraneous electron scattering by liquid water can be kept to a minimum. It still remains to be determined whether microprobe analysis (X-ray or electron energy-loss spectroscopy) using wet specimens gives better element localization in cells than the current freezing methods. More extensive comparisons are also required of the ultrastructural preservation and visibility of macromolecules immersed in a thin layer of water vs immersion in a thin layer of amorphous ice. However, the recent introduction of commercial forms of the necessary equipment now make these comparisons more feasible.     

Palaeoenvironmental reconstruction of dinosaur nesting sites based on a geochemical approach to eggshells and associated palaeosols (Maastrichtian, Provence Basin, France)

Variability of the stable isotopic (δ¹³C and δ¹⁸O) and trace element (Mg, Sr, Fe, and Mn) content from dinosaur eggshells from the Provence Basin has been addressed by considering the stratigraphic distribution of the eggshells, the palaeoenvironments, and the chemical content of associated sediments. Preferentially, in situ eggshells were analysed. Samples were collected from two sections, one belonging to the floodplain (Roques Hautes) and the other to the channel belt (Rousset-Bréguières), as determined from sedimentological criteria. On a regional scale, this combined analysis provided new insights into palaeohydrology, vegetation cover and dinosaur behaviour. Different sources of drinking water were identified: (1) the main stream waters with significant salinity levels, (2) lateral feeders with water composition close to that of precipitation. Water composition is reflected in the eggshell trace element contents: a high Sr content for the eggshells from the channel belt (mean 1200 ppm) contrasting with the very low values for the floodplain (mean 250 ppm). During the first part of the Maastrichtian, evaporation rates were quite significant, as seen from the 1.5‰ difference between the eggshell δ¹⁸O (drinking water) and the palaeosol carbonate nodules (precipitation). This contrast diminished and became nearly insignificant in the second half of the Maastrichtian. Vegetation was composed of C₃ plants and two ecosystems are reconstructed: a riparian forest extending along the channel belt and open vegetation in the floodplain. The contrasted isotopic and trace element content of the eggshells was a crucial factor in identifying animal migration from one site to another. Migration was quite limited, suggesting that the animals settled for some time at the places where they laid their eggs. Intraspecific variations among modern birds are well correlated with the environment and may be greater than interspecies variations, which limits the potential of geochemical analysis to characterise dinosaur species.     

Epitaxial crystallization of alkane chain lipids for electron diffraction analysis

Thin microcrystals of a wide variety of polymethylene chain materials, including n-alkanes, linear waxes, glycerides, a detergent, phospholipids and phospholipid analogs based on cyclopentane-1,2,3-triol, are epitaxially grown on naphthalene to give an orientation with long chain axes parallel to the best developed crystal face. These crystals, which represent a different orientation than those grown from solution, facilitate ab initio quantitative crystal structure analysis from electron diffraction intensity data from the projection yielding the most crystallographic information.     

Paleolimulus, an early limuline (Xiphosurida), from Pennsylvanian-Permian Lagerst tten of Kansas and taphonomic comparison with modern Limulus

The Pennsylvanian-Permian horseshoe crab Paleolimulus signatus (Beecher), incorporating as a junior synonym P. avitus Dunbar, is one of the earliest species of the Limulina (Xiphosurida). Some specimens from Kansas, USA, are exceptionally well preserved, retaining intact book gills and appendages. Indistinct, bilobed burrowing traces of variable width occur in association with some examples of P. signatus and may have been produced by that animal. Based on actualistic taphonomic experiments on Limulus polyphemus, ancient horseshoe crabs and other arthropods having non-mineralized exoskeletons are inferred to have become pliable soon after death or moulting, and to have disarticulated slowly prior to burial. Extreme compression, wrinkling, and loose folding of sclerites are attributed to burial of a pliable exoskeleton. Slow preburial disarticulation partly accounts for the exceptional preservation of Paleolimulus remains. Also relevant for the exceptional preservation of these arthropods was burial in estuarine, tidal flat, or lacustrine environments. Because of fluctuating salinity and possibly dessicating conditions, these settings were limiting to scavengers, burrowers, and some microbes that could potentially disarticulate or decompose xiphosurid remains.     

Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED

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Decay of vertebrate characters in hagfish and lamprey (Cyclostomata) and the implications for the vertebrate fossil record

The timing and sequence of events underlying the origin and early evolution of vertebrates remains poorly understood. The palaeontological evidence should shed light on these issues, but difficulties in interpretation of the non-biomineralized fossil record make this problematic. Here we present an experimental analysis of decay of vertebrate characters based on the extant jawless vertebrates (Lampetra and Myxine). This provides a framework for the interpretation of the anatomy of soft-bodied fossil vertebrates and putative cyclostomes, and a context for reading the fossil record of non-biomineralized vertebrate characters. Decay results in transformation and non-random loss of characters. In both lamprey and hagfish, different types of cartilage decay at different rates, resulting in taphonomic bias towards loss of 'soft' cartilages containing vertebrate-specific Col2α1 extracellular matrix proteins; phylogenetically informative soft-tissue characters decay before more plesiomorphic characters. As such, synapomorphic decay bias, previously recognized in early chordates, is more pervasive, and needs to be taken into account when interpreting the anatomy of any non-biomineralized fossil vertebrate, such as Haikouichthys, Mayomyzon and Hardistiella.     

Preliminary X-ray diffraction analysis of crystals of turnip yellow mosaic virus (TYMV)

Turnip yellow mosaic virus (TYMV) was purified from Chinese cabbage and crystallized in a form that permits high resolution structural analysis using X-ray diffraction. The crystals have a hexagonal bipyramidal morphology and often achieve dimensions of 1.0 × 1.0 × 0.5 mm. The crystals appear to be of hexagonal space group P6₂22 with a = b = 525 Å, c=315 Å, but we cannot strictly rule out the possibility that the space group is P622. They appear different than any crystals of TYMV previously reported. There are three T = 3 virus particles in the unit cell, which implies that one quarter of the particle, or 45 protein subunits, comprises the asymmetric unit of the crystal. Native data have been collected using synchrotron radiation to a resolution of 3.2 Å. © 1995 Wiley-Liss, Inc.     

Basal dinosauriform and theropod dinosaurs from the mid–late Norian (Late Triassic) of Poland: implications for Triassic dinosaur evolution and distribution

The rise of dinosaurs during the Triassic is a widely studied evolutionary radiation, but there are still many unanswered questions about early dinosaur evolution and biogeography that are hampered by an unevenly sampled Late Triassic fossil record. Although very common in western North America and parts of South America, dinosaur (and more basal dinosauriform) remains are relatively rare in the Upper Triassic deposits of Europe, making any new discoveries critically important. One of the most diverse dinosauriform assemblages from Europe comes from the Por?ba site in Poland, a recently described locality with exposures of the Zb?szynek Beds, which have a palynomorph assemblage characteristic for the mid–late Norian in the biostratigraphic schemes of the Germanic Basin. Using a synapomorphy‐based approach, we evaluate several isolated dinosauriform specimens from Por?ba. This assemblage includes a silesaurid, a herrerasaurid and remains of another type of theropod (potentially a neotheropod). The Por?ba herrerasaurid is the first record of this rare group of primitive dinosaurs from Europe and one of the youngest records worldwide, whereas the silesaurid is the youngest record of a silesaurid from Europe. These findings indicate that silesaurids persisted alongside true dinosaurs into the mid–late Norian of Europe and that silesaurid–herrerasaurid–neotheropod assemblages (which are also known from the Norian of North America, at low latitudes) were more widespread geographically and latitudinally than previously thought. Silesaurid–herrerasaurid–neotheropod assemblages may have been a common ecological structuring of dinosaurs during their early evolution, and their widespread distribution may indicate weak palaeolatitudinal controls on early dinosaur biogeography during the latest Triassic.     

The skull of Monolophosaurus jiangi (Dinosauria: Theropoda) and its implications for early theropod phylogeny and evolution

The Middle Jurassic was a critical time in the evolution of theropod dinosaurs, highlighted by the origination and initial radiation of the large‐bodied and morphologically diverse Tetanurae. Middle Jurassic tetanurans are rare, but have been described from Europe, South America and China. In particular, China has yielded a number of potential basal tetanurans, but these have received little detailed treatment in the literature. Chief among these is Monolophosaurus jiangi, known from a single skeleton that includes a nearly complete and well‐preserved skull characterized by a bizarre cranial crest. Here, we redescribe the skull of Monolophosaurus, which is one of the most complete basal tetanuran skulls known and the only quality source of cranial data for Middle Jurassic Chinese theropods. The cranial crest is atomized into a number of autapomorphic features and several characters confirm the tetanuran affinities of Monolophosaurus. However, several features suggest a basal position within Tetanurae, which contrasts with most published cladistic analyses, which place Monolophosaurus within the more derived Allosauroidea. Cranial characters previously used to diagnose Allosauroidea are reviewed and most are found to have a much wider distribution among Theropoda, eroding an allosauroid position for Monolophosaurus and questioning allosauroid monophyly. The use of phylogenetic characters relating to theropod cranial crests is discussed and a protocol for future use is given. The systematic position of Guanlong wucaii is reviewed, and a basal tyrannosauroid affinity is upheld contrary to one suggestion of a close relationship between this taxon and Monolophosaurus. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 573–607.     

Purification, crystallization and preliminary crystallographic investigations of selenium-containing phycocyanin from selenium-rich algae (Spirulina platensis)

The selenium-containing phycocyanin from the selenium-rich algae (Spirulina platensis) has been crystallized in two crystal forms by the hanging-drop vapor diffusion techniques. A chromatographic procedure of gel filtration and anion exchange was used for purification. Form I crystal with space group P2₁ and cell parameters a =108.0 Å , b = 117.0 Å , c = 184.0 Å , β = 90.2° and 12(αβ ) units in the asymmetric unit was obtained by using (NH₄)₂SO₄ as precipitant. These crystals diffract up to 2.8 Å . Form II crystal obtained by using PEG4000 as precipitant belongs to space group P63 with unit cell constants a = 155.0 Å , c = 40.3 Å , γ =120.0° and one(αβ ) unit in the asymmetric unit. The crystals diffract beyond 2.9 Å . The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.     

A general purpose package (MADFOMS) for predicting the morphologies and powder X-ray diffraction patterns of molecular crystals

We present a generalised procedure for predicting the morphologies and diffraction patterns of molecular crystals. The general purpose program MADFOMSs (Morphologies and Diffraction For Organic Molecular Solids) is described. The success of the approach is illustrated by application to the polymorphs of 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile.     

Taphonomy of modern communal burrow systems of the Plains vizcacha (Lagostomus maximus,Chinchillidae) in the Pampas region of Argentina: implications for the fossil record

The Plains vizcacha (Lagostomus maximus) is one of the largest rodents in South America. They live in communal burrow systems (vizcacheras) shaped by complex subterranean galleries which produce a strong impact on the local landscape. This paper presents the results of an actualistic study conducted with abandoned vizcacheras from the Pampas region of Argentina. The main objective is to evaluate the role of this rodent in the formation of the fossil record. Results indicate that the Plains vizcacha is responsible for the mixing, accumulation, and transport of materials; such as sticks, caliche, dung, feces, and abundant bone remains. Their burrowing activity and the accumulating habits, modifies the landscape, creating environments conducive to the buildup of objects and the reuse by different animals. These characteristics result in very complex associations of materials of different origins; making this rodent an important taphonomic agent with the potential to significantly impact the fossil record.     

Comparative spermatozoal ultrastructure and molecular analysis in dromiid crabs and their phylogenetic implications for Dromiidae and Podotremata (Decapoda: Brachyura)

We described the spermatozoal ultrastructure and conducted a molecular analysis of Dromiidae Hypoconcha parasitica, Hypoconcha arcuata, Moreiradromia antillensis and Dromia erythropus. To elucidate the relationship between the different species of this brachyuran group, we also compared the spermatozoal morphologies and phylogenetic positioning among species of Dromiidae, Dromioidea and Podotremata. Specimens were collected from the northern coast of São Paulo, Brazil and were fixed and processed followed by transmission electron microscopy and molecular analysis routines. The Dromiidae spermatozoa studied are characterized by a discoidal acrosome, with three or four concentric zones, which are centrally separated by a bilaterally capitate perforatorial chamber, with a “mushroom”-shaped apex in the Hypoconchinae and a “T-shape” in Dromiinae. Above the perforatorial chamber, there is an apical protuberance, continuous with the subopercular region and the operculum, which forms a low, centrally perforated dome. Under differential interference contrast microscopy, the spermatozoa show 3 to 4 radial arms. The spermatozoal characters in Hypoconchinae and Dromiinae do not separate these subfamilies from the Dromiidae and Dromioidea. Ultrastructural differentiation was only found between representative Dromioidea and other Podotremata. Thus, the spermiotaxonomy of these Hypoconcha, Moreiradromia and Dromia species corroborated previous morphological and molecular studies, supporting the monophyly of Dromiidae and Dynomenidae in relation to Homolidae and Latreilliidae.     

The composition of the spikes ofAcanthosphaera zachariasi (Chlorococcales): Investigations with fluorescent lectins and with X-ray diffraction

Summary The spikes ofAcanthosphaera zachariasi (Chlorococcales) were tested for lectin binding, staining reactions, solubility in trifluoroacetic acid (TFA) and with X-ray diffraction. The matrix stains only with Concanavalin A, not with different other lectins. The TFA resistant part is shown in negative stain to consist of ribbon-like fibrils with kinking sites. The spikes reveal a cellulose I-like X-ray diffraction pattern. It is concluded that they consist of cellulose associated with non-cellulosic matrix components.     

Hydration properties of N-(alpha-hydroxyacyl)-sphingosine: X-ray powder diffraction and FT-Raman spectroscopic studies

The thermotropic properties of N-(alpha-hydroxyacyl)-sphingosine (CER[AS]) in dry and hydrated state were studied by means of X-ray powder diffraction and FT-Raman spectroscopy. The polymorphic states of the CER[AS]/water mixture (lamellar crystalline, lamellar hexagonal gel, liquid crystalline) depend on the thermal pre-treatment of the sample. Only by heating the CER[AS]/water mixture above the melting chain transition can the system be hydrated. At room temperature, both dry and hydrated states form lamellar structures, which differ in their repeat distance and packing of hydrocarbon chains. Above the melting chain transition, hydrated CER[AS] forms a liquid crystalline hexagonal phase, whereas anhydrous CER[AS] forms an isotropic liquid phase. The various phases of hydrated CER[AS] are distinguished on the basis of the corresponding Raman spectra.