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.     

Crystalline structure,orientation and nucleation of the nacreous tablets in the cephalopod <Emphasis Type="Italic">Nautilus</Emphasis>

The nacreous tablets in the Nautilus shell have similar crystalline structure as the tablets in the gastropod Gibbula shell. Etching with Mutvei’s solution reveals that each tablet is composed of vertical crystalline columns that are structurally similar to the acicular crystallites in the outer spherulitic-prismatic layer of the shell wall. The columns are attached to each other to form numerous vertical crystalline lamellae, oriented parallel to the longitudinal axis of the tablet. It is still unknown whether or not the orientation of the vertical lamellae corresponds to that of the crystallographic a- or b-axis. The orientation of the crystalline lamellae in the adjacent tablets is parallel in some nacreous laminae, but random in other laminae. Similar large variation was found in the nacreous tablets of the gastropod and bivalve shells. The nucleation sites of the nacreous tablets are predominantly situated on the peripheral portion of the upper surface of the preceding tablet, both in the shell wall and septa. The “aragonite-nucleating proteins” in the central portion of the crystal imprints of the organic interlamellar sheets, described by several writers, have therefore a negative correlation with the nucleation sites of the nacreous tablets.     

Preliminary assessment of the use of electron backscatter diffraction (EBSD) in conodonts

Pérez‐Huerta, A., Cusack, M. & Méndez, C.A. 2011: Preliminary assessment of the use of electron backscatter diffraction (EBSD) in conodonts. Lethaia, Vol. 45, pp. 253–258. Conodonts are extensively used as ‘geochemical tools’ in palaeoenvironmental reconstructions in Palaeozoic and Triassic strata. The retention of a primary geochemical signal is substantially dependent on the ultrastructural integrity and crystallinity after diagenesis. Direct assessments of the preservation potential of the polycrystalline matrix are scarce with the exception of the application of transmission electron microscopy (TEM). The possible application of electron backscatter diffraction (EBSD) is introduced for well‐preserved Pennsylvanian platform conodont elements as a novel in situ technique based on successful previous results for biogenic carbonates. EBSD shows the presence of no diffraction in central regions of all studied elements of Idiognathodus and Adetognathus, with possible implications for geochemistry analysis. The absence of diffraction could be attributed to the existence of an amorphous phase, water (+OH) or a process of hypocalcification related to the development of ‘growth cavities’ in the lamellar crown tissue. Overall results indicate the suitability of using EBSD to extract crystallographic information from conodonts and potentially for other apatite (micro‐) fossils. □Conodont, diagenesis, EBSD, geochemistry, Pennsylvanian.     

A brachiopod boring (Podichnus) in a Late Cretaceous oyster from a mangrove-like environment, Skåne, Sweden

A characteristic etching trace, comparable to those produced by pedicles of some Recent brachiopods, was found on a Late Cretaceous, Campanian oyster shell from a mangrove-like setting in the Åsen quarry, Sweden. The trace fossil belongs to the ichnospecies Podichnus centrifugalis Bromley & Surlyk, 1973 and is, together with a single specimen of Crania craniolaris Linnæus, 1758, the only evidence of brachiopods in the mangrove-like environment.     

Multiscale structure of calcite fibres of the shell of the brachiopod Terebratulina retusa

The shells of rhynchonelliform brachiopods have an outer (primary) layer of acicular calcite and an inner (secondary) layer of calcite fibres which are parallel to the shell exterior. Atomic force microscopy (AFM) reveals that these fibres are composed of large triangular nanogranules of about 600-650 nm along their long axis. The nanogranules are composites of organic and inorganic components. As the shell grows, the fibres elongate with the calcite c-axis perpendicular to the fibre axis as demonstrated by electron backscatter diffraction (EBSD). Thus, despite being a composite structure comprising granules that are themselves composites, each fibre is effectively a single crystal. The combination of AFM and EBSD reveals the details of the structure and crystallography of these fibres. This knowledge serves to identify those aspects of biological control that must be understood to enable comprehension of the biological control exerted on the construction of these exquisite biomineral structures.     

Diagenetic fate of bioapatite in linguliform brachiopods: multiple apatite phases in shells of Cambrian lingulate brachiopod Ungula ingrica (Eichwald)

Fossil skeletal apatites vary in their composition and can yield mixed biochemical, environmental and diagenetic information. Thus, it is important to evaluate the diagenesis spatially inside the skeleton. We study the cross sections of shells of the Furongian lingulate brachiopod Ungula ingrica from Estonia using the Attenuated Total Reflectance – Fourier Transform Infrared (ATR‐FTIR) microspectroscopic and energy dispersive spectroscopic (EDS) mapping and show for the first time that different structural laminae of the shell have different chemical compositions. Compact laminae are rich in PO₄^3?, Na, Mg and poor in F and Ca. Porous (baculate) laminae are rich in carbonate anions, Ca and F, but contain less Na and Mg. The ATR‐FTIR spectra show further differences in the ν₂ carbonate region, where the IR band at 872 cm^?1 in compact laminae is replaced by a strong band at 864 cm^?1 in baculate laminae. The changes in shell apatite suggest different origins of the apatite phases. Compact laminae are likely chemically less altered and could potentially carry more reliable palaeoenvironmental or geochemical information than the apatite in baculate laminae, which is mostly authigenic in its origin.     

Evolutionary and Diagenetic Changes in the Chemico-structure of the Shell of Cranioid Brachiopods

The laminar dorsal valve of living Neocrania consists of: a primary layer of rhombohedral tablets, composed of granular calcite and commonly forming slats orthogonal to the margin, associated with polysaccharides and a fibrous 60 kDa protein; and a secondary layer of spirally growing (10.4) rhombohedra, doped with the 60 kDa protein and interleaved with membranes of a fibrous 44 kDa protein. The ventral valve consists exclusively of a primary layer with the same composition and basic structure as that of the dorsal valve. Investigation of selected antecedents shows that the chemico-structure of the Neocrania shell has been virtually unchanged since the first appearance of the stock in the Early Ordovician (Arenig). The greatest phylogenetic change affected the ventral valve that varied, even in Ordovician genera, from a film of calcitic blades to a complete succession of primary and secondary laminae. The most profound diagenetic changes occurred before the Late Cretaceous with proteins degrading into peptides that were dispersed, with the loss of less stable amino acids, during laminar recrystallization. Palaeozoic shells suffered further recrystallization but, even after pressure solution, the original laminar fabric was replicated long after it had lost its constraining organic membranes.     

Characterization of the calcium biomineral in the radular teeth of Chiton pelliserpentis

The radula in a group of molluscan invertebrates, the chitons (Polyplacophora), is a ribbon-like apparatus used for feeding and which bears a series of distinctive mineralized teeth called the major lateral teeth. While some chiton species deposit only iron biominerals in these teeth, many others deposit both iron and calcium. In this study, the calcium biomineral in the teeth of one of the latter types of species, the Australian east-coast chiton, Chiton pelliserpentis, has been isolated and examined for the first time. Spectroscopic and crystallographic techniques have identified the biomineral as a carbonate-substituted apatite with significant fluoride substitution also likely. Fourier-transform infrared and laser Raman spectroscopy indicated that the carbonate content was less than that of either bovine tibia cortical bone or human tooth enamel. X-ray diffraction analysis showed the biomineral to be poorly crystalline due to small crystal size and appreciable anionic substitution. The lattice parameters were calculated to be a=9.382?Å and c=6.883?Å, which are suggestive of a fluorapatite material. It is postulated that structural and biochemical differences in the tooth organic matrix of different chiton species will ultimately determine if the teeth become partly calcified or iron mineralized only.     

CYTOPLASMIC MASSES PRESERVED IN EARLY HOLOCENE DIATOMS: A POSSIBLE TAPHONOMIC PROCESS AND ITS PALEO‐ECOLOGICAL IMPLICATIONS1

In Lake Suigetsu, central Japan, greenish/light‐brown granules identified as cytoplasmic masses had been preserved in siliceous cell walls of freshwater diatoms in annual layers of lacustrine muds since the early Holocene. The lacustrine muds consisted of alternating dark‐colored (rich in diatom valves, clay, and organic matter) and light‐colored (mainly diatom valves) laminae. The greenish/light‐brown granules were predominately preserved in frustules of the genus Aulacoseira preserved in the dark‐colored laminae. The dark‐colored laminae were inferred to have formed annually under stratified water caused by surface water warming in summer that caused the formation of an organic‐rich anoxic layer on the lake bottom that favored granule preservation. The good preservation of cytoplasmic masses in dark‐colored laminae suggested a cause for diatom assemblage periodicity, a phenomenon that was commonly noted in temperate lakes: the cells containing these masses could be potential seed stocks for subsequent spring blooms. Frustules of the most abundant granule‐containing species, Aulacoseira nipponica (Skvortzow) Tuji, in the dark‐colored laminae of the Early Holocene muds were abundant in the overlying light‐colored laminae, suggesting that these species reproduced abundantly in springtime yielding a massive diatom bloom.     

Electron backscatter diffraction (EBSD) analysis of maniraptoran eggshells with important implications for microstructural and taphonomic interpretations

Electron backscatter diffraction (EBSD) is a useful tool for gathering crystallographic information from carbonate biominerals because it maps out the orientation of crystal grains very precisely. EBSD has become popular in invertebrate palaeontology but its application in vertebrate palaeontology remains limited. However, the study of fossil eggshells is a field where EBSD has wide potential applicability and provides a quantitative approach to fossil eggshell research as well as new qualitative data. Here we analyse fossil and extant maniraptoran dinosaur eggshells using EBSD analysis emphasizing four different aspects. The mapping imaging clarifies previously ambiguous characters such as squamatic ultrastructure and allows a more objective evaluation of avian and non‐avian maniraptoran eggshell. In particular, our results imply that the presence of an external zone in the manirpatoran eggshell is not diagnostic of avian eggshell. EBSD analysis can be also used for differentiating true pore canals from cracks in the eggshell radial section, thereby determining the biological genuineness or otherwise of a pore‐like structure. Finally, the misorientation angle distribution of the material shows a clear dichotomy that may reflect reproductive brooding strategy, although further studies on contact incubation of palaeognaths and neognaths are needed to confirm this.     

Hierarchically structured scleractinian coral biocrystals

Microscopic (AFM and FESEM) observations show that scleractinian coral biomineral fibers in extant Desmophyllum and Favia, and fossil Jurassic Isastrea are composed of nanocrystalline grains of about 30-100 nm in size. In contrast to these findings, SR diffraction data on the same coral materials exhibit narrow Bragg peaks suggesting much larger crystallite size. These seemingly contradicting results of microscopic and diffraction studies are reconciled within a new, minute-scale model of scleractinian biomineral fibers. In this model, nanocrystalline aragonite units are interconnected by mineral bridges and form aggregates usually larger than 200 nm. Most likely, the size of the aggregates is resulting from physiological biomineralization cycles that control cellular secretion of ions and biopolymeric species. Intercalation of biopolymers into crystal lattice may influence consistently several structural parameters of the scleractinian coral bio-aragonite in all studied samples: (i) the lattice parameters and internal strains of the bio-aragonite are larger than in mineral aragonite, (ii) lattice parameter elongations and internal strains reveal directional anisotropy with respect to crystallographic axes.     

Continental-scale tree population response to rapid climate change, competition and disturbance

Aim  Using a new approach to analyse fossil pollen data, we investigate temporal and spatial patterns in Populus ( poplar, cottonwood, aspen) from the Late Glacial to the present at regional to continental scales.
Location  North America.
Methods  We extracted data on the timing and magnitude of the maximum value of Populus pollen from each pollen diagram in the North American Pollen Database (NAPD). The information was plotted in histograms of 150-year bins to identify times when Populus was abundant on the landscape. We also mapped the maximum values to identify spatial patterns and their causes.
Results  Our analyses show that there have been several periods since the Late Glacial when Populus was abundant on the landscape: (1) from 12.35 to 12.65 kyr  bp , in eastern North America, largely in response to the opening of the forest following the onset of the Younger Dryas; (2) from 10.85 to 11.75 kyr  bp , following the termination of the Younger Dryas; and (3) during the last 150 years, as land was cleared for agricultural use, especially in the midwestern United States.
Main conclusion  Since the Late Glacial, changes in the abundance of Populus were caused more by the effects of abrupt climate change on its major competitors, rather than the direct effects of climate on Populus itself.     

Fossil evidence and phylogeography of temperate species: 'glacial refugia' and post-glacial recolonization

We present a short synthesis of the Pleistocene distribution dynamics and phylogeographic recolonization hypotheses for two temperate European mammal species, the red deer ( Cervus elaphus ) and the roe deer ( Capreolus capreolus ), for which high-resolution patterns of fossil evidence and genetic data sets are available. Such data are critical to an understanding of the role of hypothesized glacial refugia. Both species show a similar pattern: a relatively wide distribution in the southern part of Central Europe 60,000–25,000 years ago, and a strong restriction to areas in southern Europe for nearly 10,000 years during the Last Glacial Maximum (LGM) and the early Late Glacial (25,000–14,700 years ago). With the beginning of Greenland Interstadial 1 (Bølling/Allerød warming, c. 14,700–11,600 years ago) a sudden range expansion into Central Europe is visible, but the colonization of most of Central Europe, including the northern European Lowlands, only began in the early Holocene. In a European context, regions where the species were distributed during the LGM and early Late Glacial are most relevant as potential origins of recolonization processes, because during these c. 10,000 years distribution ranges were smaller than at any other time in the Late Quaternary. As far as the present distribution of temperate species and their genetic lineages is concerned, so-called 'cryptic refugia' are important only if the species are actually confirmed there during the LGM, as otherwise they could not possibly have contributed to the recolonization that eventually resulted in the present distribution ranges.     

Chemico-structural phylogeny of the discinoid brachiopod shell

Stratiform shells of living discinids are composed of membranous laminae and variously aggregated, protein-coated granules of apatitic francolite supported by proteinaceous and chitinous nets in glycosaminoglycans (GAGs) to form laminae in rhythmic sets. The succession is like that of living lingulids but differs significantly in the structure of the periostracum, the nature of baculate sets and in its organic composition. In particular, discinids have a higher level of amino acids although with relatively lower acidic and higher basic concentrations; and their overall lower organic content is owing to lower levels of hydrophilic components, like GAGs and chitin. The organic constituents are not completely degraded during fossilization; but data are presently too meagre to distinguish between linguloid and discinoid ancestries. Many differences among three of the four described extant genera emanate from transformations with a long geological history. Pelagodiscus is characterized by regular, concentric rheomorphic folding (fila) of the flexible periostracum and the plastic primary layer and by sporadically developed hemispherical imprints of periostracal vesicles. Both features are more strikingly developed in Palaeozoic discinids. In the oldest discinid, the Ordovician Schizotreta, and the younger Orbiculoidea and related genera, vesicles were persistent, hexagonal close-packed arrays fading out over fila. They must have differed in composition, however, as the larger vesicles of Schizotreta were simple (possibly mucinous), whereas the smaller vesicles of Orbiculoidea and younger genera were composites of thickly coated spheroids, possibly of lipoproteins, which survive as disaggregated relicts in Pelagodiscus. Baculate sets within the secondary layer are also less well developed in living discinids, being incipient in Pelagodiscus and restricted to the dorsal valve of Discinisca. The trellised rods (baculi) with proteinaceous cores are composed of pinacoids or prisms of apatite, depending on whether they are supported by chitinous nets or proteinaceous strands in GAGs. This differentiation occurred in Schizotreta but in that stock (and Trematis) the baculate set is symmetrical with baculi subtended between compact laminae, whereas in younger and post-Palaeozoic species the outer bounding lamina(e) of the set is normally membranous and/or stratified. The most striking synapomorphy of living discinids is the intravesicular secretion of organsiliceous tablets with a crystalline habit within the larval outer epithelium and their exocytosis as a close- or open-packed, transient, biomineral cover for larvae. Canals, on the other hand, are homologous with those pervading lingulid shells. Both systems interconnect with chitinous and proteinaceous sets and have probably always served as vertical struts in an organic scaffolding supporting the stratiform successions. A phylogenetic analysis based mainly on shell structure confirms the discinoids as the sister group of the linguloids but, contrary to current taxonomic practice, also supports the inclusion of acrotretoids within a ''discinoid'' clade as a sister group to the discinids.     

A non-destructive technique for chemical mapping of insect inclusions in amber

Synchrotron-based techniques offer a wealth of elemental, molecular, and structural insights in biological samples, but the application of these techniques to fossils is a relatively new development. Here we examine how synchrotron radiation micro X-ray fluorescence (SR µXRF) may be used to investigate the chemical composition of insects trapped in amber, while leaving the inclusions unaltered. Elemental distribution data could provide important information on tissue preservation in insect inclusions, as well as information on the processes involved in fossilization. By analyzing a series of ants (Hymenoptera: Formicidae) that range from modern material, to Eocene Baltic amber, and Late Cretaceous North Carolina amber, we investigate how variable preservation influences the results obtained through SR µXRF analyses, as well as the various merits and pitfalls associated with the application of this technique to amber inclusions. This work serves as an introduction to the underlying principles, strengths, and limitations associated with applying SR µXRF in a palaeontological context.     

UBERABATITAN RIBEIROI, A NEW TITANOSAUR FROM THE MARÍLIA FORMATION (BAURU GROUP, UPPER CRETACEOUS), MINAS GERAIS, BRAZIL

Abstract:  A new Late Cretaceous titanosaur sauropod from the Bauru Basin of Brazil, Uberabatitan ribeiroi gen. et sp. nov., represented by at least three specimens, is described. The material comes from a level of coarse sandstone within the Serra da Galga sequence in Uberaba County, Minas Gerais State. The fossiliferous strata belong to the Marília Formation (Serra da Galga Member), Bauru Group, considered to be Maastrichtian in age. The fossils occur in the uppermost levels of the above-mentioned unit; thus, Uberabatitan ribeiroi is the youngest titanosaur to have been recorded from the Bauru Basin. The autapomorphies that support the new species are: (1) anterior and mid-cervicals with postzygodiapophyseal lamina (podl) segmented in zygapophyseal and diapophyseal laminae, of which the first extends rostrodorsally over the second; (2) mid-dorsals with a robust lateral lamina formed mainly by a diapophyseal lamina (probably homologous to the postzygodiapophyseal lamina), and, to a lesser extent, by a relic of the spinodiapophyseal lamina (spdl); (3) mid (and probably posterior) dorsals with accessory neural laminae, which are lateral to the prespinal lamina, and probably homologous to the spinoprezygapophyseal laminae (sprl); (4) mid-caudal centra with deeply excavated lateral faces; (5) pubis very thick and robust, with a very stout longitudinal crest on its external (ventral) face; and (6) proximal end of the tibia with a prominent lateral protuberance, which articulates with an equally prominent medial knob of the fibula. The titanosaurian assemblage at Uberaba includes, apart from U. ribeiroi , well-preserved specimens assigned to species of uncertain affinities ( Trigonosaurus pricei and Baurutitan britoi ), as well as a few vertebrae assigned to aeolosaurines.     

Growth of protein-doped rhombohedra in the calcitic shell of craniid brachiopods

The secondary shell of the living inarticulated brachiopod Neocrania consists of calcitic laminae interleaved with organic sheets, predominantly a 44 kDa protein with high levels of aspartic acid–asparagine and glutamic acid–glutamine. Laminae consist of tabular (01.4) rhombohedra that are composed of spherular or rhombohedral granules, ca. 30 nm in size. Rhombohedra increase by planar or spiral growth as granular, monolayered plates that commonly act as foundations for multilayered tablets up to 300 nm or so high. Rough (0k.l) faces, kinked by cleavage, usually develop at either end of the long diagonals of rhombohedra, the edges of which may support ramparts that can accrete centripetally to enclose any organic material situated at tablet centres. Induced degradation by proteinase K shows that sectors subtended by (0k.l) steps are doped by a fibrous polymer, identified as an exclusively intralaminar glycosylated 60 kDa protein. A 44 kDa protein has also been extracted from laminae and is presumably incorporated into tablets by centripetally growing ramparts.     

A NEW PIPINE FROG FROM AN EOCENE CRATER LAKE IN NORTH-CENTRAL TANZANIA

Abstract:  The fossiliferous site of Mahenge, Tanzania, interpreted as a small lake that formed in a kimberlite intrusion, has yielded a vertebrate assemblage that includes scarce frog remains. Radiometric dating of the pipe indicates that the volcanic activity took place at 45·83 ± 0·17 Ma, whereas infilling of the crater was completed in 0·2–1·0 myr after emplacement. The frogs, described herein, are preserved as partially articulated skeletons that represent a new pipine taxon, Singidella latecostata gen. et sp. nov. A parsimony analysis of fossil and extant pipoid frogs indicates that Singidella is more closely related to African Hymenochirus and Pseudhymenochirus than to South American Pipa . It also provides weak support for the sister-group relationship of Singidella and the bizarre Late Cretaceous Pachycentrata taqueti (replacement name for Pachybatrachus taqueti ) from Niger, although the incomplete preservation of Pachycentrata precludes thorough comparisons. This record indicates that prior to the rifting in the Miocene the distribution of pipines extended into eastern Africa where they do not occur today.     

Ecological impacts of a widespread frost event following early spring leaf‐out

In the spring of 2010, temperatures averaged ~3 °C above the long‐term mean (March–May) across the northeastern United States. However, in mid‐to‐late spring, much of this region experienced a severe frost event. The spring of 2010 therefore provides a case study on how future spring temperature extremes may affect northeastern forest ecosystems. We assessed the response of three northern hardwood tree species (sugar maple, American beech, yellow birch) to these anomalous temperature patterns using several different data sources and addressed four main questions: (1) Along an elevational gradient, how was each species affected by the late spring frost? (2) How did differences in phenological growth strategy influence their response? (3) How did the late spring frost affect ecosystem productivity within the study domain? (4) What are the potential long‐term impacts of spring frost events on forest community ecology? Our results show that all species exhibited early leaf development triggered by the warm spring. However, yellow birch and American beech have more conservative growth strategies and were largely unaffected by the late spring frost. In contrast, sugar maples responded strongly to warmer temperatures and experienced widespread frost damage that resulted in leaf loss and delayed canopy development. Late spring frost events may therefore provide a competitive advantage for yellow birch and American beech at the expense of sugar maple. Results from satellite remote sensing confirm that frost damage was widespread throughout the region at higher elevations (>500 m). The frost event is estimated to have reduced gross ecosystem productivity by 70–153 g C m^?2, or 7–14% of the annual gross productivity (1061 ± 82 g C m^?2) across 8753 km² of high‐elevation forest. We conclude that frost events following leaf out, which are expected to become more common with climate change, may influence both forest composition and ecosystem productivity.