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Placental structure is linked to function across morphological scales. In the placenta, changes to gross anatomy, such as surface area, volume, or blood vessel arrangement, are associated with suboptimal physiological outcomes. However, quantifying each of these metrics requires different laborious semi-quantitative methods. Here, we demonstrate how, with minimal sample preparation, whole-organ computed microtomography (microCT) can be used to calculate gross morphometry of the equine placenta and a range of additional metrics, including branching morphometry of placental vasculature, non-destructively from a single dataset. Our approach can be applied to quantify the gross structure of any large mammalian placenta., Competing Interests: Declaration of competing interest We declare we have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We use synchrotron x-ray tomography of annual growth increments in the dental cementum of mammaliaforms (stem and crown fossil mammals) from three faunas across the Jurassic to map the origin of patterns of mammalian growth patterns, which are intrinsically related to mammalian endothermy. Although all fossils studied exhibited slower growth rates, longer life spans, and delayed sexual maturity relative to comparably sized extant mammals, the earliest crown mammals developed significantly faster growth rates in early life that reduced at sexual maturity, compared to stem mammaliaforms. Estimation of basal metabolic rates (BMRs) suggests that some fossil crown mammals had BMRs approaching the lowest rates of extant mammals. We suggest that mammalian growth patterns first evolved during their mid-Jurassic adaptive radiation, although growth remained slower than in extant mammals.

Mammalian placentas exhibit unparalleled structural diversity, despite sharing a common ancestor and principal functions. The bulk of structural studies in placental research has used two-dimensional (2D) histology sectioning, allowing significant advances in our understanding of mammalian placental structure. However, 2D histology sectioning may be limited if it does not provide accurate information of three-dimensional (3D) tissue architecture. Here, we propose correlative 3D X-ray histology (3D-XRH) as a tool with great potential for resolving mammalian placental structures. 3D-XRH involves scanning a formaldehyde-fixed, paraffin embedded (FFPE) tissue block with 3D X-ray microscopy (microCT) prior to histological sectioning to generate a 3D image volume of the embedded tissue piece. The subsequent 2D histology sections can then be correlated back into the microCT image volume to couple histology staining (or immunolabelling) with 3D tissue architecture. 3D-XRH is non-destructive and requires no additional sample preparation than standard FFPE histology sectioning, however the image volume provides 3D morphometric data and can be used to guide microtomy. As such, 3D-XRH introduces additional information to standard histological workflows with minimal effort or disruption. Using primary examples from porcine, bovine, equine, and canine placental samples, we demonstrate the application of 3D-XRH to quantifying placental structure as well as discussing the limitations and future directions of the methodology. The wealth of information derived from 2D histological sectioning in the biomedical, veterinary, and comparative reproductive sciences provides a rich foundation from which 3D-XRH can build on to advance the study of placental structure and function., Competing Interests: Declaration of competing interest We declare we have no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Despite having a single evolutionary origin and conserved function, the mammalian placenta exhibits radical structural diversity. The evolutionary drivers and functional consequences of placental structural diversity are poorly understood. Humans and equids both display treelike placental villi, however these villi evolved independently and exhibit starkly different levels of invasiveness into maternal tissue (i.e. the number of maternal tissue layers between placental tissue and maternal blood). The villi in these species therefore serve as a compelling evolutionary case study to explore whether placentas have developed structural adaptations to respond to the challenge of reduced nutrient availability in less invasive placentas. Here, we use three-dimensional X-ray microfocus computed tomography and electron microscopy to quantitatively evaluate key structures involved in exchange in human and equid placental villi. We find that equid villi have a higher surface area to volume ratio and deeper trophoblastic vessel indentation than human villi. Using illustrative computational models, we propose that these structural adaptations have evolved in equids to boost nutrient transfer to compensate for reduced invasiveness into maternal tissue. We discuss these findings in relation to the 'maternal-fetal conflict hypothesis' of placental evolution.

The placenta mediates physiological exchange between the mother and the fetus. In placental mammals, all placentas are descended from a single common ancestor and functions are conserved across species; however, the placenta exhibits radical structural diversity. The selective pressures behind this structural diversity are poorly understood. Traditionally, placental structures have largely been investigated by grouping them into qualitative categories. Assessing the placenta on this basis could be problematic when inferring the relative "efficiency" of a placental configuration to transfer nutrients from mother to fetus. We argue that only by considering placentas as three-dimensional (3D) biological structures, integrated across scales, can the evolutionary questions behind their enormous structural diversity be quantitatively determined. We review the current state of placental evolution from a structural perspective, detail where 3D imaging and computational modeling have been used to gain insight into placental function, and outline an experimental roadmap to answer evolutionary questions from a multiscale 3D structural perspective. Our approach aims to shed light on placental evolution, and can be transferred to evolutionary investigations in any organ system., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE).)

The digital reconstruction of neurocranial endocasts has elucidated the gross brain structure and potential ecological attributes of many fossil taxa, including Irritator, a spinosaurine spinosaurid from the "mid" Cretaceous (Aptian) of Brazil. With unexceptional hearing capabilities, this taxon was inferred to integrate rapid and controlled pitch-down movements of the head that perhaps aided in the predation of small and agile prey such as fish. However, the neuroanatomy of baryonychine spinosaurids remains to be described, and potentially informs on the condition of early spinosaurids. Using micro-computed tomographic scanning (μCT), we reconstruct the braincase endocasts of Baryonyx walkeri and Ceratosuchops inferodios from the Wealden Supergroup (Lower Cretaceous) of England. We show that the gross endocranial morphology is similar to other non-maniraptoriform theropods, and corroborates previous observations of overall endocranial conservatism amongst more basal theropods. Several differences of unknown taxonomic utility are noted between the pair. Baryonychine neurosensory capabilities include low-frequency hearing and unexceptional olfaction, whilst the differing morphology of the floccular lobe tentatively suggests less developed gaze stabilisation mechanisms relative to spinosaurines. Given the morphological similarities observed with other basal tetanurans, baryonychines likely possessed comparable behavioural sophistication, suggesting that the transition from terrestrial hypercarnivorous ancestors to semi-aquatic "generalists" during the evolution of Spinosauridae did not require substantial modification of the brain and sensory systems., (© 2023 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Isolated spinosaurid teeth are relatively well represented in the Lower Cretaceous Wealden Supergroup of southern England, UK. Until recently it was assumed that these teeth were referable to Baryonyx , the type species ( B. walkeri ) and specimen of which is from the Barremian Upper Weald Clay Formation of Surrey. British spinosaurid teeth are known from formations that span much of the c. 25 Ma depositional history of the Wealden Supergroup, and recent works suggest that British spinosaurids were more taxonomically diverse than previously thought. On the basis of both arguments, it is appropriate to doubt the hypothesis that isolated teeth from outside the Upper Weald Clay Formation are referable to Baryonyx . Here, we use phylogenetic, discriminant and cluster analyses to test whether an isolated spinosaurid tooth (HASMG G369a, consisting of a crown and part of the root) from a non-Weald Clay Formation unit can be referred to Baryonyx . HASMG G369a was recovered from an uncertain Lower Cretaceous locality in East Sussex but is probably from a Valanginian exposure of the Hastings Group and among the oldest spinosaurid material known from the UK. Spinosaurid affinities are both quantitatively and qualitatively supported, and HASMG G369a does not associate with Baryonyx in any analysis. This supports recent reinterpretations of the diversity of spinosaurid in the Early Cretaceous of Britain, which appears to have been populated by multiple spinosaurid lineages in a manner comparable to coeval Iberian deposits. This work also reviews the British and global records of early spinosaurids (known mainly from dental specimens), and revisits evidence for post-Cenomanian spinosaurid persistence., Competing Interests: The authors declare there are no competing interests., (©2023 Barker et al.)

Three-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Age-related changes in bone microstructure can inform our understanding the biology of both extant and fossil birds, but to date, histological work in birds, and particularly work using high-resolution 3D imaging, has largely been restricted to limited growth stages. We used minimally destructive synchrotron radiation-based X-ray computed tomography to visualise and measure key morphological and histological traits in 3D across development in the domestic duck and ring-necked pheasant. We use these measurements to build on the database of key reference material for interpreting bone histology. We found that growth patterns differed between the two species, with the ducks showing rapid growth in their lower limbs and early lower limb maturation, while pheasants grew more slowly, reflecting their later age at maturity. In the pheasant, both walking and flight occur early and their upper and lower limbs grew at similar rates. In the duck, flight and wing development are delayed until the bird is almost at full body mass. Through juvenile development, the second moment of area for the duck wing was low but increased rapidly towards the age of flight, at which point it became significantly greater than that of the lower limb, or the pheasant. On a microstructural level, both cortical porosity and canal diameter were related to cortical bone deposition rate. In terms of orientation, vascular canals in the bone cortex were more laminar in the humerus and femur compared with the tibiotarsus, and laminarity increased through juvenile development in the humerus, but not the tibiotarsus, possibly reflecting torsional vs compressive loading. These age-related changes in cortical bone vascular microstructure of the domestic duck and pheasant will help understanding the biology of both extant and fossil birds, including age estimation, growth rate and growth patterns, and limb function., (© 2022 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Postcranial elements (cervical, sacral and caudal vertebrae, as well as ilium, rib and limb bone fragments) belonging to a gigantic tetanuran theropod were recovered from the basal unit (the White Rock Sandstone equivalent) of the Vectis Formation near Compton Chine, on the southwest coast of the Isle of Wight. These remains appear to pertain to the same individual, with enormous dimensions similar to those of the Spinosaurus holotype and exceeding those of the largest European theropods previously reported. A combination of features-including the presence of spinodiapophyseal webbing on an anterior caudal vertebra-suggest that this is a member of Spinosauridae, though a lack of convincing autapomorphies precludes the identification of a new taxon. Phylogenetic analysis supports spinosaurid affinities but we were unable to determine a more precise position within the clade weak support for a position within Spinosaurinae or an early-diverging position within Spinosauridae were found in some data runs. Bioerosion in the form of curved tubes is evident on several pieces, potentially related to harvesting behaviour by coleopteran bioeroders. This is the first spinosaurid reported from the Vectis Formation and the youngest British material referred to the clade. This Vectis Formation spinosaurid is unusual in that the majority of dinosaurs from the Lower Cretaceous units of the Wealden Supergroup are from the fluviolacustrine deposits of the underlying Barremian Wessex Formation. In contrast, the lagoonal facies of the upper Barremian-lower Aptian Vectis Formation only rarely yield dinosaur material. Our conclusions are in keeping with previous studies that emphasise western Europe as a pivotal region within spinosaurid origination and diversification., Competing Interests: The authors declare there are no competing interests., (©2022 Barker et al.)

Cementum, the tissue attaching mammal tooth roots to the periodontal ligament, grows appositionally throughout life, displaying a series of circum-annual incremental features. These have been studied for decades as a direct record of chronological lifespan. The majority of previous studies on cementum have used traditional thin-section histological methods to image and analyse increments. However, several caveats have been raised in terms of studying cementum increments in thin-sections. Firstly, the limited number of thin-sections and the two-dimensional perspective they impart provide an incomplete interpretation of cementum structure, and studies often struggle or fail to overcome complications in increment patterns that complicate or inhibit increment counting. Increments have been repeatedly shown to both split and coalesce, creating accessory increments that can bias increment counts. Secondly, identification and counting of cementum increments using human vision is subjective, and it has led to inaccurate readings in several experiments studying individuals of known age. Here, we have attempted to optimise a recently introduced imaging modality for cementum imaging; X-ray propagation-based phase-contrast imaging (PPCI). X-ray PPCI was performed for a sample of rhesus macaque (Macaca mulatta) lower first molars (n = 10) from a laboratory population of known age. PPCI allowed the qualitative identification of primary/annual versus intermittent secondary increments formed by splitting/coalescence. A new method for semi-automatic increment counting was then integrated into a purpose-built software package for studying cementum increments, to count increments in regions with minimal complications. Qualitative comparison with data from conventional cementochronology, based on histological examination of tissue thin-sections, confirmed that X-ray PPCI reliably and non-destructively records cementum increments (given the appropriate preparation of specimens prior to X-ray imaging). Validation of the increment counting algorithm suggests that it is robust and provides accurate estimates of increment counts. In summary, we show that our new increment counting method has the potential to overcome caveats of conventional cementochronology approaches, when used to analyse three-dimensional images provided by X-ray PPCI., Competing Interests: The authors have read the journal’s policy and have the following competing interests: The authors received material support in the form of travel to the Swiss Light Source Synchrotron for our experiment, consisting of airfare, guesthouse reservations, and external hard drives. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Travel to the Swiss Light Source Synchrotron for our experiment, consisting of airfare, guesthouse reservations, and external hard drives.

Spinosaurids are among the most distinctive and yet poorly-known of large-bodied theropod dinosaurs, a situation exacerbated by their mostly fragmentary fossil record and competing views regarding their palaeobiology. Here, we report two new Early Cretaceous spinosaurid specimens from the Wessex Formation (Barremian) of the Isle of Wight. Large-scale phylogenetic analyses using parsimony and Bayesian techniques recover the pair in a new clade within Baryonychinae that also includes the hypodigm of the African spinosaurid Suchomimus. Both specimens represent distinct and novel taxa, herein named Ceratosuchops inferodios gen. et sp. nov. and Riparovenator milnerae gen. et sp. nov. A palaeogeographic reconstruction suggests a European origin for Spinosauridae, with at least two dispersal events into Africa. These new finds provide welcome information on poorly sampled areas of spinosaurid anatomy, suggest that sympatry was present and potentially common in baryonychines and spinosaurids as a whole, and contribute to updated palaeobiogeographic reconstructions for the clade., (© 2021. The Author(s).)

Take-off is a critical phase of flight, and many birds jump to take to the air. Although the actuation of the hindlimb in terrestrial birds is not limited to the sagittal plane, and considerable non-sagittal plane motion has been observed during take-off jumps, how the spatial arrangement of hindlimb muscles in flying birds facilitates such jumps has received little attention. This study aims to ascertain the 3D hip muscle function in the magpie ( Pica pica ), a bird known to jump to take-off. A musculoskeletal model of the magpie hindlimb was developed using μCT scans (isotropic resolution of 18.2 μm) to derive bone surfaces, while the 3D muscle path definition was further informed by the literature. Function was robustly characterized by determining the 3D moment-generating capacity of 14 hip muscles over the functional joint range of motion during a take-off leap considering variations across the attachment areas and uncertainty in dynamic muscle geometry. Ratios of peak flexion-extension (FE) to internal-external rotation (IER) and abduction-adduction (ABD) moment-generating capacity were indicators of muscle function. Analyses of 972 variations of the 3D muscle paths showed that 11 of 14 muscles can act as either flexor or extensor, while all 14 muscles demonstrated the capacity to act as internal or external rotators of the hip with the mean ratios of peak FE to IER and ABD moment-generating capacity were 0.89 and 0.31, respectively. Moment-generating capacity in IER approaching levels in the FE moment-generating capacity determined here underline that the avian hip muscle function is not limited to the sagittal plane. Together with previous findings on the 3D nature of hindlimb kinematics, our results suggest that musculoskeletal models to develop a more detailed understanding of how birds orchestrate the use of muscles during a take-off jump cannot be restricted to the sagittal plane., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Meilak, Gostling, Palmer and Heller.)

Cementum is a mineralized dental tissue common to mammals that grows throughout life, following a seasonally appositional rhythm. Each year, one thick translucent increment and one thin opaque increment is deposited, offering a near-complete record of an animal's life history. Male and female mammals exhibit significant differences in oral health, due to the contrasting effects of female versus male sex hormones. Oestrogen and progesterone have a range of negative effects on oral health that extends to the periodontium and cementum growth interface. Here, we use synchrotron radiation-based X-ray tomography to image the cementum of a sample of rhesus macaque ( Macaca mulatta ) teeth from individuals of known life history. We found that increased breeding history in females corresponds with increased increment tortuosity and less organized cementum structure, when compared to male and juvenile cementum. We quantified structural differences by measuring the greyscale 'texture' of cementum and comparing results using principal components analysis. Adult females and males occupy discrete regions of texture space with no overlap. Females with known pregnancy records also have significantly different cementum when compared with non-breeding and juvenile females. We conclude that several aspects of cementum structure and texture may reflect differences in sexual life history in primates.

Despite considerable advances in knowledge of the anatomy, ecology and evolution of early mammals, far less is known about their physiology. Evidence is contradictory concerning the timing and fossil groups in which mammalian endothermy arose. To determine the state of metabolic evolution in two of the earliest stem-mammals, the Early Jurassic Morganucodon and Kuehneotherium, we use separate proxies for basal and maximum metabolic rate. Here we report, using synchrotron X-ray tomographic imaging of incremental tooth cementum, that they had maximum lifespans considerably longer than comparably sized living mammals, but similar to those of reptiles, and so they likely had reptilian-level basal metabolic rates. Measurements of femoral nutrient foramina show Morganucodon had blood flow rates intermediate between living mammals and reptiles, suggesting maximum metabolic rates increased evolutionarily before basal metabolic rates. Stem mammals lacked the elevated endothermic metabolism of living mammals, highlighting the mosaic nature of mammalian physiological evolution.

Describing and quantifying vascular canal orientation and volume of osteocyte lacunae in bone is important in studies of bone growth, mechanics, health and disease. It is also an important element in analysing fossil bone in palaeohistology, key to understanding the growth, life and death of extinct animals. Often, bone microstructure is studied using two-dimensional (2D) sections, and three-dimensional (3D) shape and orientation of structures are estimated by modelling the structures using idealised geometries based on information from their cross sections. However, these methods rely on structures meeting strict geometric assumptions. Recently, 3D methods have been proposed which could provide a more accurate and robust approach to bone histology, but these have not been tested in direct comparison with their 2D counterparts in terms of accuracy and sensitivity to deviations from model assumptions. We compared 2D and 3D methodologies for estimating key microstructural traits using a combination of experimental and idealised test data sets. We generated populations of cylinders (canals) and ellipsoids (osteocyte lacunae), varying the cross-sectional aspect ratios of cylinders and orientation of ellipsoids to test sensitivity to deviations from cylindricality and longitudinal orientation, respectively. Using published methods, based on 2D sections and 3D data sets, we estimated cylinder orientation and ellipsoid volume. We applied the same methods to six CT data sets of duck cortical bone, using the full volumes for 3D measurements and single CT slices to represent 2D sections. Using in silico test data sets that did deviate from ideal cylinders and ellipsoids resulted in inaccurate estimates of cylinder or canal orientation, and reduced accuracy in estimates of ellipsoid and lacunar volume. These results highlight the importance of using appropriate 3D imaging and quantitative methods for quantifying volume and orientation of 3D structures and offer approaches to significantly enhance our understanding of bone physiology based on accurate measures for bone microstructures., (© 2020 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

The use of in vivo X-ray microcomputed tomography (μCT) to study plant root systems has become routine, but is often hampered by poor contrast between roots, soil, soil water, and soil organic matter. In clinical radiology, imaging of poorly contrasting regions is frequently aided by the use of radio-opaque contrast media. In this study, we present evidence for the utility of iodinated contrast media (ICM) in the study of plant root systems using μCT. Different dilutions of an ionic and nonionic ICM (Gastrografin 370 and Niopam 300) were perfused into the aerial vasculature of juvenile pea plants via a leaf flap (Pisum sativum). The root systems were imaged via μCT, and a variety of image-processing approaches used to quantify and compare the magnitude of the contrast enhancement between different regions. Though the treatment did not appear to significantly aid extraction of full root system architectures from the surrounding soil, it did allow the xylem and phloem units of seminal roots and the vascular morphology within rhizobial nodules to be clearly visualized. The nonionic, low-osmolality contrast agent Niopam appeared to be well tolerated by the plant, whereas Gastrografin showed evidence of toxicity. In summary, the use of iodine-based contrast media allows usually poorly contrasting root structures to be visualized nondestructively using X-ray μCT. In particular, the vascular structures of roots and rhizobial nodules can be clearly visualized in situ.

The origin and radiation of mammals are key events in the history of life, with fossils placing the origin at 220 million years ago, in the Late Triassic period. The earliest mammals, representing the first 50 million years of their evolution and including the most basal taxa, are widely considered to be generalized insectivores. This implies that the first phase of the mammalian radiation--associated with the appearance in the fossil record of important innovations such as heterodont dentition, diphyodonty and the dentary-squamosal jaw joint--was decoupled from ecomorphological diversification. Finds of exceptionally complete specimens of later Mesozoic mammals have revealed greater ecomorphological diversity than previously suspected, including adaptations for swimming, burrowing, digging and even gliding, but such well-preserved fossils of earlier mammals do not exist, and robust analysis of their ecomorphological diversity has previously been lacking. Here we present the results of an integrated analysis, using synchrotron X-ray tomography and analyses of biomechanics, finite element models and tooth microwear textures. We find significant differences in function and dietary ecology between two of the earliest mammaliaform taxa, Morganucodon and Kuehneotherium--taxa that are central to the debate on mammalian evolution. Morganucodon possessed comparatively more forceful and robust jaws and consumed 'harder' prey, comparable to extant small-bodied mammals that eat considerable amounts of coleopterans. Kuehneotherium ingested a diet comparable to extant mixed feeders and specialists on 'soft' prey such as lepidopterans. Our results reveal previously hidden trophic specialization at the base of the mammalian radiation; hence even the earliest mammaliaforms were beginning to diversify--morphologically, functionally and ecologically. In contrast to the prevailing view, this pattern suggests that lineage splitting during the earliest stages of mammalian evolution was associated with ecomorphological specialization and niche partitioning.

· Root hairs are known to be highly important for uptake of sparingly soluble nutrients, particularly in nutrient deficient soils. Development of increasingly sophisticated mathematical models has allowed uptake characteristics to be quantified. However, modelling has been constrained by a lack of methods for imaging live root hairs growing in real soils. · We developed a plant growth protocol and used Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM) to uncover the three-dimensional (3D) interactions of root hairs in real soil. We developed a model of phosphate uptake by root hairs based directly on the geometry of hairs and associated soil pores as revealed by imaging. · Previous modelling studies found that root hairs dominate phosphate uptake. By contrast, our study suggests that hairs and roots contribute equally. We show that uptake by hairs is more localized than by roots and strongly dependent on root hair and aggregate orientation. · The ability to image hair-soil interactions enables a step change in modelling approaches, allowing a more realistic treatment of processes at the scale of individual root hairs in soil pores., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Abundant charcoalified seed fern (pteridosperm) pollen organs and ovules have been recovered from Late Viséan (Mississippian 330 Ma) limestones from Kingswood, Fife, Scotland. To overcome limitations of data collection from these tiny, sometimes unique, fossils, we have combined low vacuum scanning electron microscopy on uncoated specimens with backscatter detector and synchrotron radiation X-ray tomographic microscopy utilizing the Materials Science and TOMCAT beamlines at the Swiss Light Source of the Paul Scherrer Institut. In combination these techniques improve upon traditional cellulose acetate peel sectioning because they enable study of external morphology and internal anatomy in multiple planes of section on a single specimen that is retained intact. The pollen organ Melissiotheca shows a basal parenchymatous cushion bearing more than 100 sporangia on the distal face. Digital sections show the occurrence of pollen in some sporangia. The described ovule is new and has eight integumentary lobes that are covered in spirally arranged glandular hairs. Virtual longitudinal sections reveal the lobes are free above the pollen chamber. Results are applied in taxonomy and will subsequently contribute to our understanding of the former diversity and evolution of ovules, seeds, and pollen organs in the seed ferns, the first seed-bearing plants to conquer the land.

Experimental analyses of decay in a tunicate deuterostome and three lophotrochozoans indicate that the controls on decay and preservation of embryos, identified previously based on echinoids, are more generally applicable. Four stages of decay are identified regardless of the environment of death and decay. Embryos decay rapidly in oxic and anoxic conditions, although the gross morphology of embryos is maintained for longer under anoxic conditions. Under anoxic reducing conditions, the gross morphology of the embryos is maintained for the longest period of time, compatible with the timescale required for bacterially mediated mineralization of soft tissues. All four stages of decay were encountered under all environmental conditions, matching the spectrum of preservational qualities encountered in all fossil embryo assemblages. The preservation potential of embryos of deuterostomes and lophotrochozoans is at odds with the lack of such embryos in the fossil record. Rather, the fossil record of embryos, as sparse as it is, is dominated by forms interpreted as ecdysozoans, cnidarians, and stem-metazoans. The dearth of deuterostome and lophotrochozoan embryos may be explained by the fact that ecdysozoans, at least, tend to deposit their eggs in the sediment rather than through broadcast spawning. However, fossil embryos remain very rare and the main controlling factor on their fossilization may be the unique conspiracy of environmental conditions at a couple of sites. The preponderance of fossilized embryos of direct developers should not be used in evidence against the existence of indirect development at this time in animal evolutionary history.

Stereoblastic embryos from the Doushantuo Formation of China exhibit occasional asynchronous cell division, with diminishing blastomere volume as cleavage proceeded. Asynchronous cell division is common in modern embryos, implying that sophisticated mechanisms for differential cell division timing and embryonic cell lineage differentiation evolved before 551 million years ago. Subcellular structures akin to organelles, coated yolk granules, or lipid vesicles occur in these embryos. Paired reniform structures within embryo cells may represent fossil evidence of cells about to undergo division. Embryos exhibit no evidence of epithelial organization, even in embryos composed of approximately 1000 cells. Many of these features are compatible with metazoans, but the absence of epithelialization is consistent only with a stem-metazoan affinity for Doushantuo embryos.

Fossilized embryos from the late Neoproterozoic and earliest Phanerozoic have caused much excitement because they preserve the earliest stages of embryology of animals that represent the initial diversification of metazoans. However, the potential of this material has not been fully realized because of reliance on traditional, non-destructive methods that allow analysis of exposed surfaces only, and destructive methods that preserve only a single two-dimensional view of the interior of the specimen. Here, we have applied synchrotron-radiation X-ray tomographic microscopy (SRXTM), obtaining complete three-dimensional recordings at submicrometre resolution. The embryos are preserved by early diagenetic impregnation and encrustation with calcium phosphate, and differences in X-ray attenuation provide information about the distribution of these two diagenetic phases. Three-dimensional visualization of blastomere arrangement and diagenetic cement in cleavage embryos resolves outstanding questions about their nature, including the identity of the columnar blastomeres. The anterior and posterior anatomy of embryos of the bilaterian worm-like Markuelia confirms its position as a scalidophoran, providing new insights into body-plan assembly among constituent phyla. The structure of the developing germ band in another bilaterian, Pseudooides, indicates a unique mode of germ-band development. SRXTM provides a method of non-invasive analysis that rivals the resolution achieved even by destructive methods, probing the very limits of fossilization and providing insight into embryology during the emergence of metazoan phyla.

The vertebrate Zic gene family encodes C2H2 zinc finger transcription factors closely related to the Gli proteins. Zic genes are expressed in multiple areas of developing vertebrate embryos, including the dorsal neural tube where they act as potent neural crest inducers. Here we describe the characterization of a Zic ortholog from the amphioxus Branchiostoma floridae and further describe the expression of a Zic ortholog from the ascidian Ciona intestinalis. Molecular phylogenetic analysis and sequence comparisons suggest the gene duplications that formed the vertebrate Zic family were specific to the vertebrate lineage. In Ciona maternal CiZic/Ci-macho1 transcripts are localized during cleavage stages by asymmetric cell division, whereas zygotic expression by neural plate cells commences during neurulation. The amphioxus Zic ortholog AmphiZic is expressed in dorsal mesoderm and ectoderm during gastrulation, before being eliminated first from midline cells and then from all neurectoderm during neurulation. After neurulation, expression is reactivated in the dorsal neural tube and dorsolateral somite. Comparison of CiZic and AmphiZic expression with vertebrate Zic expression leads to two main conclusions. First, Zic expression allows us to define homologous compartments between vertebrate and amphioxus somites, showing primitive subdivision of vertebrate segmented mesoderm. Second, we show that neural Zic expression is a chordate synapomorphy, whereas the precise pattern of neural expression has evolved differently on the different chordate lineages. Based on these observations we suggest that a change in Zic regulation, specifically the evolution of a dorsal neural expression domain in vertebrate neurulae, was an important step in the evolution of the neural crest.