The Moscovian staffellids from Ziyun County,Guizhou Province,China: the effects of sedimentary environment on fusulinid taxonomic discrepancy

Carboniferous fusulinids, including 23 species in 7 genera, are identified from the Lumazhai area, Ziyun County, southern Guizhou, China. They are mainly composed of Staffella, Pseudoendothyra, Pseudostaffella, and Fusulinella, which suggest a late Moscovian age. The fusulinid assemblage is characterized by the dominance of staffellids that shows a different composition from the other late Moscovian fusulinid faunas reported in South China. Combined with microfacies analysis, we suggest that the different faunal composition most likely resulted from varying depositional settings, implying the sensitivity of fusulinids to sedimentary environments. This phenomenon of “synchronous but heterogeneous fusulinid biofacies”, caused by changing sedimentary condition, is significant in explaining taxonomic variation especially with limited collection of fusulinid fossils.     

Palaeopathology of the Pleistocene specimen D2600 from Dmanisi (Republic of Georgia)

Here we present a detailed palaeopathological study of the hominin mandible D2600 recovered at the Dmanisi site, Republic of Georgia. The Dmanisi assemblage represents the earliest evidence of hominins outside Africa with an age of 1.8 Ma. D2600 is the holotype of Homo georgicus species and its taxonomic assignment is still under debate. Our study reveals severe and unusual dental wear accompanied of extensive root exposure and dental axial migration, periapical abscesses and enamel fractures. In addition, there is evidence of post-eruptive tooth rotation and temporomandibular arthropathy. We propose that the wear pattern observed in this individual is related to a diet with a high intake of fibrous and abrasive foods such as fruits and plants, as it is usually recorded in chimpanzees and gorillas and unlike the wear pattern observed in other Homo specimens of our comparative sample. The rounded occlusal surfaces and highly polished labio-lingual surfaces of D2600 anterior teeth could be mainly the consequence of pre- and/or para-masticatory activities such as gripping and stripping. This type of food would be also the origin of the highly cupped occlusal morphology of the posterior dentition in combination with relatively slight approximal attrition. However, the lesions exhibited by D2600 have not significantly altered the morphology of the mandible and do not prevent a proper taxonomic assessment.     

Magnetic resonance microscopy of prenatal dolphins (Mammalia, Odontoceti, Delphinidae) - Ontogenetic and phylogenetic implications

The structure of two preserved prenatal dolphins were visualized by 3D MR microscopy (isotropic nominal resolution up to 78.1 μm), which is a high-resolution 3D magnetic resonance imaging (MRI) technique. To determine the benefits and limitations of this method, the acquired 3D datasets were segmented manually and compared to histological sections of different specimens in corresponding developmental stages. The MR images visualize the external and internal morphology of both prenatal dolphins in detail. Various organ systems with their main components are clearly documented in the images, allowing a complete segmentation of the specimens and the calculation of volumes and surface areas of different organ systems. Due to its non-invasive character and the detailed imaging within its resolution range, MR microscopy proves to be a valuable tool in developmental biology for the visualization of the inner architecture of rare and delicate museum specimens, such as the small dolphin embryo and fetus examined. In these two prenatal dolphins, the profound structural modifications at the transition from the embryonic to the fetal stage reflect the adaptations of the mammalian bauplan to the requirements of a holaquatic cetacean life-style. However, the developmental pattern and sequence of the emerging tissues and organs in prenatal life do not resemble the hypothesized evolution of the structural and functional adaptations found in the fossil record.     

Redefinition and taxonomic revision of the “buqueti” species-group,Dichotomius Hope, 1838 (Coleoptera: Scarabaeidae: Scarabaeinae)

On the basis of external morphology and genitalia of males, as well as a comparison between different species belonging to other groups of Dichotomius, we propose a redefinition of the “buqueti” species group separated by Luederwaldt (1929). Six species are excluded from this group and transferred to other groups: D. ribeiroi (Pereira, 1954) in the “cotopaxi” group; D. camposeabrai Martínez, 1974 in the “bitiensis” group; D. reclinatus (Felsche, 1901), D. horridus (Felsche, 1911), D. quadrinodosus (Felsche, 1901) and D. nimuendaju (Luederwaldt, 1925) (revalidated species) forming the newly defined “reclinatus” group. The other species kept in the “buqueti” group include D. buqueti (Lucas, 1857) from Brazil (lectotype here designated), D. haroldi (Waterhouse, 1891) from Argentina and D. nutans (Harold, 1867) from Brazil, Argentina and Uruguay. The taxonomic revision of the “buqueti” group are presented, including a history of the included species, a determination key, illustrations of the structures of external morphology, male genitalia and sclerites of the internal sac, and a distribution map.     

Baring it all: undressing Cambrian ‘Orsten’ phosphatocopine crustaceans using synchrotron radiation X‐ray tomographic microscopy

Synchrotron radiation X‐ray tomographic microscopy (SRXTM) was used to virtually dissect and peel the shields off of the microscopic, bivalved phosphatocopine crustaceans in the Cambrian ‘Orsten’ type of preservation of Sweden. Doing so opened up for an array of concealed internal structures to be observed in a fully enclosed specimen of Hesslandona ventrospinata and a semi‐enclosed specimen of Hesslandona angustata. For comparison, also a head‐larva stage specimen of H. angustata, with shields in ‘butterfly position’, was analysed. The X‐ray tomographic data sets revealed excellently preserved structures, such as labrum, sternum, antennae, mandibular and post‐mandibular limbs with their minute setae, all of which were more or less disguised by the enclosing shields. This, moreover, allowed assignment to growth stages of the specimens, which is impossible based solely on external morphology and size. Micro‐spherules observed inside the shields of the semi‐enclosed H. angustata specimen may represent remains of food particles, and the feeding biology of phosphatocopines is discussed in detail. Our analyses suggest that phosphatocopines were particle feeders. The SRXTM technique offers the ability to three‐dimensionally reconstruct the morphology in high resolution, construct virtual serial sections and study concealed structures. The resulting data allow for new structures to be revealed for previously known taxa and for new taxa to be identified, with the added benefit of not destroying the specimens in the process. Hence, we do not longer have to rely on serendipitous finds of broken and/or open phosphatocopine specimens to elucidate their diagnostic ventral morphology.     

It’s not what it looks like: molecular data fails to substantiate morphological differences in two sea hares (Mollusca,Heterobranchia, Aplysiidae) from southern Brazil

Species of sea hares have been recognized traditionally based on morphological traits, mainly the radula, external coloration, and reproductive anatomy. However, recent studies have shown substantial color variation in some sea slug species. Molecular data have been successfully used to differentiate morphologically similar species of “opisthobranchs” and resolve questions on the taxonomic value of color. The objective of this paper is to use molecular data in an attempt to elucidate whether specimens of Aplysia brasiliana with distinct colorations and morphologies are actually the same species. To this end, DNA from 14 specimens of A. brasiliana was extracted, including five specimens identified as a distinct morphotype from typical A. brasiliana. Although the two morphotypes have consistent differences in their external morphology and radula, the molecular data confirmed that there are no significant genetic differences between them. This is another example of the need to re-evaluate taxonomic decisions based on morphology in light of molecular evidence.     

On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography

The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.     

Visualizing the 3D architecture of multiple erythrocytes infected with Plasmodium at nanoscale by focused ion beam-scanning electron microscopy

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures.     

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.     

High quality 3D imaging of vertebrate microremains using X-ray synchrotron phase contrast microtomography

Vertebrate microremains, particularly teeth, represent a substantial part of known vertebrate biodiversity. Many groups, such as Mesozoic mammals, are known mostly through isolated teeth. Classical imaging techniques of such complex millimetric to inframillime-tric objects are most often limited by problems of manipulation, depth of focus or limited orientation. The methods generally used are stereomicroscopy (including in-focus z-series reconstruction) and Scanning Electron Microscopy (SEM), which provide good images. Nevertheless, both provide 2D static images or partial and directional 3D data, making complete observation difficult. Propagation phase contrast synchrotron X-ray microtomography is a powerful technique alleviating these limitations. Thanks to submicron resolution and to the edge detection effect, it rapidly provides 3D data from minute samples with levels of quality and detail unattainable using conventional microtomographs. Complex morphology of small specimens can be studied with unlimited orientation possibilities and, when coupled with 3D printing, it allows enlarged 3D reproductions of such small and fragile fossils.     

Estimating Mass Properties of Dinosaurs Using Laser Imaging and 3D Computer Modelling

Body mass reconstructions of extinct vertebrates are most robust when complete to near-complete skeletons allow the reconstruction of either physical or digital models. Digital models are most efficient in terms of time and cost, and provide the facility to infinitely modify model properties non-destructively, such that sensitivity analyses can be conducted to quantify the effect of the many unknown parameters involved in reconstructions of extinct animals. In this study we use laser scanning (LiDAR) and computer modelling methods to create a range of 3D mass models of five specimens of non-avian dinosaur; two near-complete specimens of Tyrannosaurus rex, the most complete specimens of Acrocanthosaurus atokensis and Strutiomimum sedens, and a near-complete skeleton of a sub-adult Edmontosaurus annectens. LiDAR scanning allows a full mounted skeleton to be imaged resulting in a detailed 3D model in which each bone retains its spatial position and articulation. This provides a high resolution skeletal framework around which the body cavity and internal organs such as lungs and air sacs can be reconstructed. This has allowed calculation of body segment masses, centres of mass and moments or inertia for each animal. However, any soft tissue reconstruction of an extinct taxon inevitably represents a best estimate model with an unknown level of accuracy. We have therefore conducted an extensive sensitivity analysis in which the volumes of body segments and respiratory organs were varied in an attempt to constrain the likely maximum plausible range of mass parameters for each animal. Our results provide wide ranges in actual mass and inertial values, emphasizing the high level of uncertainty inevitable in such reconstructions. However, our sensitivity analysis consistently places the centre of mass well below and in front of hip joint in each animal, regardless of the chosen combination of body and respiratory structure volumes. These results emphasize that future biomechanical assessments of extinct taxa should be preceded by a detailed investigation of the plausible range of mass properties, in which sensitivity analyses are used to identify a suite of possible values to be tested as inputs in analytical models.     

Neutron microtomography‐based virtual extraction and analysis of a cercopithecoid partial cranium (STS 1039) embedded in a breccia fragment from sterkfontein member 4 (South Africa)

The Plio‐Pleistocene karstic sedimentary deposits of Sterkfontein Cave, South Africa, yielded numerous fossil primate specimens embedded in blocks of indurated breccia, including the partial cercopithecoid cranium labelled STS 1039. Because the surrounding matrix masks most of its morphology, the specimen remains taxonomically undetermined. While the use of X‐ray microtomography did not allow extracting any structural information about the specimen, we experimented a new investigative technique based on neutron microtomography. Using this innovative approach, we successfully virtually extracted, reconstructed in 3D and quantitatively assessed the preserved dentognathic structural morphology of STS 1039, including details of its postcanine maxillary dentition. Following comparative analyses with a number of Plio‐Pleistocene and extant cercopithecoid taxa, we tentatively propose a taxonomic attribution to the taxon Cercopithecoides williamsi. Our experience highlights the remarkable potential of this novel imaging method to extract diagnostic information and to identify the fossil remains embedded in hard breccia from the South African hominin‐bearing cave sites. Am J Phys Anthropol 159:737–745, 2016. © 2015 Wiley Periodicals, Inc.     

High resolution scanning electron microscopy of plant chromosomes

A preparation technique for high resolution field emission scanning electron microscopy of plant chromosomes is described. The technique was optimized to use standard squash preparations of mitotic and meiotic chromosomes from root tips of barley, wheat, and rye. After light microscopic observation and documentation, the same object can be investigated with a 100-fold higher resolution using a field emission scanning electron microscope. Tilting of the specimens provides a three-dimensional insight into chromosomal structures at different stages of condensation and decondensation. With this technique it was possible to document for the first time at high resolution structures such as the centromeric region, the spindle apparatus and the spindle fibre attachment region. The smallest unit of the DNA packed that can be resolved is a beads on a string-like structure of the nucleofilament (10–15 nm fibre).by D. Schweizer     

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (μCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that μCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.     

The Application of Optical Coherence Tomography to Image Subsurface Tissue Structure of Antarctic Krill Euphausia superba

Many small open ocean animals, such as Antarctic krill, are an important part of marine ecosystems. To discover what will happen to animals such as krill in a changing ocean, experiments are run in aquaria where conditions can be controlled to simulate water characteristics predicted to occur in the future. The response of individual animals to changing water conditions can be hard to observe, and with current observation techniques it is very difficult to follow the progress of an individual animal through its life. Optical coherence tomography (OCT) is an optical imaging technique that allows images at high resolution to be obtained from depths up to a few millimeters inside biological specimens. It is compatible with in vivo imaging and can be used repeatedly on the same specimens. In this work, we show how OCT may be applied to post mortem krill samples and how important physiological data such as shell thickness and estimates of organ volume can be obtained. Using OCT we find an average value for the thickness of krill exoskeleton to be (30±4) µm along a 1 cm length of the animal body. We also show that the technique may be used to provide detailed imagery of the internal structure of a pleopod joint and provide an estimate for the heart volume of (0.73±0.03) mm³.     

Application of molecular genetics and geometric morphometrics to taxonomy and conservation of cave beetles in central Italy

Integration of molecular genetic techniques and geometric morphometrics represent a valuable tool in the resolution of taxonomic uncertainty and the identification of significant units for conservation. We combined mitochondrial DNA cytochrome c oxidase subunit II gene sequence data and geometric morphometric analysis to examine taxonomic status and identify units for conservation in four species of the hypogean beetle Duvalius (Coleoptera, Trechinae) using mainly museum specimens collected in central Italy. Previous taxonomic studies based on morphological traits described several subspecies often inhabiting geographically distinct caves. Phylogenetic analysis identified two well supported monophyletic lineages and a number of different clades with relatively small genetic differences, suggesting a short divergence time in line with known geological history of the study area. Geometric morphometrics, on the other hand, recovered a high level of distinctiveness among specimens. Both genetic and morphometric analyses did not entirely corroborate former taxonomic nomenclature, suggesting possible rearrangements and the definition of evolutionary significant units. Beetles of the genus Duvalius are protected by regional laws and the majority of taxa considered in this study inhabit caves located outside protected areas. Our study advocates the importance of devoting protection efforts to networks of cave ecosystems rather than single locations or species.