Jaw elements in Plumulites bengtsoni confirm that machaeridians are extinct armoured scaleworms.

Machaeridians are Palaeozoic animals that are dorsally armoured with serialized, imbricating shell plates that cover or enclose the body. Prior to the discovery of an articulated plumulitid machaeridian from the Early Ordovician of Morocco that preserved unambiguous annelid characters (segmental parapodia with chaetae), machaeridians were a palaeontological mystery, having been previously linked to echinoderms, barnacles, tommotiids (putative stem-group brachiopods) or molluscs. Although the annelid affinities of machaeridians are now firmly established, their position within the phylum and relevance for understanding the early evolution of Annelida is less secure, with competing hypotheses placing Machaeridia in the stem or deeply nested within the crown group of annelids. We describe a scleritome of Plumulites bengtsoni from the Fezouata Formation of Morocco that preserves an anterior jaw apparatus consisting of at least two discrete elements that exhibit growth lines. Although jaws have multiple independent origins within the annelid crown group, comparable jaws are present only within Phyllodocida, the clade that contains modern aphroditiforms (scaleworms and relatives). Phylogenetic analysis places a monophyletic Machaeridia within the crown group of Phyllodocida in total-group Aphroditiformia, consistent with a common origin of machaeridian shell plates and scaleworm elytrae. The inclusion of machaeridians in Aphroditiformia truncates the ghost lineage of Phyllodocida by almost a hundred million years.

X-rays and virtual taphonomy resolve the first Cissus (Vitaceae) macrofossils from Africa as early-diverging members of the genus.

PREMISE OF THE STUDY: Fossilized seeds similar to Cissus (Vitaceae) have been recognized from the Miocene of Kenya, though some were previously assigned to the Menispermaceae. We undertook a comparative survey of extant African Cissus seeds to identify the fossils and consider their implications for the evolution and biogeography of Cissus and for African early Miocene paleoenvironments. METHODS: Micro-computed tomography (µCT) and synchrotron-based X-ray tomographic microscopy (SRXTM) were used to study seed morphology and anatomy. Virtual taphonomy, using SRXTM data sets, produced digital fossils to elucidate seed taphonomy. Phylogenetic relationships within Cissus were reconstructed using existing and newly produced DNA sequences for African species. Paleobiology and paleoecology were inferred from African nearest living relatives. KEY RESULTS: The fossils were assigned to four new Cissus species, related to four modern clades. The fossil plants were interpreted as climbers inhabiting a mosaic of riverine woodland and forest to more open habitats. Virtual taphonomy explained how complex mineral infill processes concealed key seed features, causing the previous taxonomic misidentification. Newly sampled African species, with seeds most similar to the fossils, belong to four clades within core Cissus, two of which are early diverging. CONCLUSIONS: Virtual taphonomy, combined with X-ray imaging, has enabled recognition of the first fossil Cissus and Vitaceae from Africa. Early-divergent members of the core Cissus clade were present in Africa by at least the early Miocene, with an African origin suggested for the Cissus sciaphila clade. The fossils provide supporting evidence for mosaic paleoenvironments inhabited by early Miocene hominoids.

Complex flow in the nasal region of guitarfishes.

Scent detection in an aquatic environment is dependent on the movement of water. We set out to determine the mechanisms for moving water through the olfactory organ of guitarfishes (Rhinobatidae, Chondrichthyes) with open nasal cavities. We found at least two. In the first mechanism, which we identified by observing dye movement in the nasal region of a life-sized physical model of the head of Rhinobatos lentiginosus mounted in a flume, olfactory flow is generated by the guitarfish's motion relative to water, e.g. when it swims. We suggest that the pressure difference responsible for motion-driven olfactory flow is caused by the guitarfish's nasal flaps, which create a region of high pressure at the incurrent nostril, and a region of low pressure in and behind the nasal cavity. Vortical structures in the nasal region associated with motion-driven flow may encourage passage of water through the nasal cavity and its sensory channels, and may also reduce the cost of swimming. The arrangement of vortical structures is reminiscent of aircraft wing vortices. In the second mechanism, which we identified by observing dye movement in the nasal regions of living specimens of Glaucostegus typus, the guitarfish's respiratory pump draws flow through the olfactory organ in a rhythmic (0.5-2 Hz), but continuous, fashion. Consequently, the respiratory pump will maintain olfactory flow whether the guitarfish is swimming or at rest. Based on our results, we propose a model for olfactory flow in guitarfishes with open nasal cavities, and suggest other neoselachians which this model might apply to.

A new fireworm (Amphinomidae) from the Cretaceous of Lebanon identified from three-dimensionally preserved myoanatomy.

BACKGROUND: Rollinschaeta myoplena gen. et sp. nov is described from the Late Cretaceous (Cenomanian) Konservat-Lagerstätten of Hakel and Hjoula, Lebanon. The myoanatomy of the fossils is preserved in exceptional detail in three dimensions as calcium phosphate, allowing the musculature of the body wall, gut and parapodia to be reconstructed in detail. RESULTS: The major muscle groups of polychaetes can be identified in Rollinschaeta, including longitudinal muscle bands, circular muscles, oblique muscles, the parapodial muscle complex and the gut musculature, with a resolution sufficient to preserve individual fibres. To allow meaningful comparison with the phosphatized fossil specimens, extant polychaetes were stained with iodine and visualised using microCT. Rollinschaeta myoplena possesses two pairs of dorsal longitudinal muscles, dorsal and ventral circular muscles and a single pair of ventral longitudinal muscles. While six longitudinal muscle bands are known from other polychaete groups, their presence in combination with circular muscles is unique to Amphinomidae, allowing these fossils to be diagnosed to family level based solely on their myoanatomy. The elongate, rectilinear body and equally sized, laterally projecting parapodia of Rollinschaeta are found only within Amphinominae, demonstrating that the Cretaceous species is derived amongst Amphinomida. CONCLUSION: The uniquely preserved myoanatomy of Rollinschaeta has allowed diagnosis of a fossil annelid to subfamily level using microCT as a comparative tool for exploring myoanatomy in fossil and extant polychaetes. Our results demonstrate that fossilized muscles can provide systematically informative anatomical detail and that they should be studied when preserved.

Cryptogamic ground covers as analogues for early terrestrial biospheres: Initiation and evolution of biologically mediated proto-soils.

Modern cryptogamic ground covers (CGCs), comprising assemblages of bryophytes (hornworts, liverworts, mosses), fungi, bacteria, lichens and algae, are thought to resemble early divergent terrestrial communities. However, limited in situ plant and other fossils in the rock record, and a lack of CGC-like soils reported in the pre-Silurian sedimentological record, have hindered understanding of the structure, composition and interactions within the earliest CGCs. A key question is how the earliest CGC-like organisms drove weathering on primordial terrestrial surfaces (regolith), leading to the early stages of soil development as proto-soils, and subsequently contributing to large-scale biogeochemical shifts in the Earth System. Here, we employed a novel qualitative, quantitative and multi-dimensional imaging approach through X-ray micro-computed tomography, scanning electron, and optical microscopy to investigate whether different combinations of modern CGC organisms from primordial-like settings in Iceland develop organism-specific soil forming features at the macro- and micro-scales. Additionally, we analysed CGCs growing on hard rocky substrates to investigate the initiation of weathering processes non-destructively in 3D. We show that thalloid CGC organisms (liverworts, hornworts) develop thin organic layers at the surface (<1 cm) with limited subsurface structural development, whereas leafy mosses and communities of mixed organisms form profiles that are thicker (up to ~ 7 cm), structurally more complex, and more organic-rich. We term these thin layers and profiles proto-soils. Component analyses from X-ray micro-computed tomography data show that thickness and structure of these proto-soils are determined by the type of colonising organism(s), suggesting that the evolution of more complex soils through the Palaeozoic may have been driven by a shift in body plan of CGC-like organisms from flattened and appressed to upright and leafy. Our results provide a framework for identifying CGC-like proto-soils in the rock record and a new proxy for understanding organism-soil interactions in ancient terrestrial biospheres and their contribution to the early stages of soil formation.

Time-lapse three-dimensional imaging of crack propagation in beetle cuticle.

Arthropod cuticle has extraordinary properties. It is very stiff and tough whilst being lightweight, yet it is made of rather ordinary constituents. This desirable combination of properties results from a hierarchical structure, but we currently have a poor understanding of how this impedes damage propagation. Here we use non-destructive, time-lapse in situ tensile testing within an X-ray nanotomography (nCT) system to visualise crack progression through dry beetle elytron (wing case) cuticle in 3D. We find that its hierarchical pseudo-orthogonal laminated microstructure exploits many extrinsic toughening mechanisms, including crack deflection, fibre and laminate pull-out and crack bridging. We highlight lessons to be learned in the design of engineering structures from the toughening methods employed. STATEMENT OF SIGNIFICANCE: We present the first comprehensive study of the damage and toughening mechanisms within arthropod cuticle in a 3D time-lapse manner, using X-ray nanotomography during crack growth. This technique allows lamina to be isolated despite being convex, which limits 2D analysis of microstructure. We report toughening mechanisms previously unobserved in unmineralised cuticle such as crack deflection, fibre and laminate pull-out and crack bridging; and provide insights into the effects of hierarchical microstructure on crack propagation. Ultimately the benefits of the hierarchical microstructure found here can not only be used to improve biomimetic design, but also helps us to understand the remarkable success of arthropods on Earth.

Micro-CT 3D imaging reveals the internal structure of three abyssal xenophyophore species (Protista, Foraminifera) from the eastern equatorial Pacific Ocean.

Xenophyophores, giant foraminifera, are distinctive members of the deep-sea megafauna that accumulate large masses of waste material ('stercomare') within their agglutinated tests, and organise their cells as branching strands enclosed within an organic tube (the 'granellare' system). Using non-destructive, three-dimensional micro-CT imaging we explored these structures in three species from the abyssal eastern Pacific Clarion-Clipperton Zone (CCZ). In Psammina spp., the low-density stercomare occupied much of the test interior, while high-density granellare strands branched throughout the structure. In Galatheammina sp. the test comprised a mixture of stercomare and test particles, with the granellare forming a web-like system of filaments. The granellare occupied 2.8-5.1%, the stercomare 72.4-82.4%, and test particles 14.7-22.5%, of the 'body' volume in the two Psammina species. The corresponding proportions in Galatheammina sp. were 1.7% (granellare), 39.5% (stercomare) and 58.8% (test particles). These data provide a potential basis for estimating the contribution of xenophyophores to seafloor biomass in areas like the CCZ where they dominate the megafauna. As in most xenophyophore species, the granellare hosted huge numbers of tiny barite crystals. We speculate that these help to support the extensive granellare system, as well as reducing the cell volume and lightening the metabolic burden required to maintain it.

Comparison of thermally actuated retro-diels-alder release groups for nanoparticle based nucleic acid delivery.

The present study explores alternate pericyclic chemistries for tethering amine-terminal biomolecules onto silver nanoparticles. Employing the versatile tool of the retro-Diels-Alder (rDA) reaction, three thermally-labile cycloadducts are constructed that cleave at variable temperature ranges. While the reaction between furan and maleimide has widely been reported, the current study also evaluates the reverse reaction kinetics between thiophene-maleimide, and pyrrole-maleimide cycloadducts. Density Functional Theorem (DFT) calculations used to model and plan the experiments, predict energy barriers for the thiophene-maleimide reverse reaction to be greatest, and the pyrrole-maleimide barriers the lowest. Based on the computational analyses, it is projected that the cycloreversion rate would occur slowest with the thiophene, followed by furan, and finally pyrrole would yield the promptest release. These thermally-responsive linkers, characterized by Electrospray Ionization Mass Spectrometry, 1H and 13C NMR, are thiol-linked to silver nanoparticles and conjugate single stranded siRNA mimics with 5' fluorescein tag. Second harmonic generation spectroscopy (SHG) and fluorescence spectroscopy are used to measure release and rate of release. The SHG decay constants and fluorescence release profiles obtained for the three rDA reactions confirm the trends obtained from the DFT computations.

A quantitative comparison of micro-CT preparations in Dipteran flies.

X-ray-based 3D-imaging techniques have gained fundamental significance in research areas ranging from taxonomy to bioengineering. There is demand for the characterisation of species-specific morphological adaptations, micro-CT (µCT) being the method of choice in small-scale animals. This has driven the development of suitable staining techniques to improve absorption-based tissue contrast. A quantitative account on the limits of current staining protocols for preparing µCT specimen, however, is still missing. Here we present a study that quantifies results obtained by combining a variety of different contrast agents and fixative treatments that provides general guidance for µCT applications, particularly suitable for insect species. Using a blowfly model system (Calliphora), we enhanced effective spatial resolution and, in particular, optimised tissue contrast enabling semi-automated segmentation of soft and hard tissue from µCT data. We introduce a novel probabilistic measure of the contrast between tissues: PTC. Our results show that a strong iodine solution provides the greatest overall increase in tissue contrast, however phosphotungstic acid offers better inter-tissue discriminability. We further show that using paraformaldehyde as a fixative as opposed to ethanol, slows down the uptake of a staining solution by approximately a factor of two.

Motion-driven flow in an unusual piscine nasal region.

Fishes have several means of moving water to effect odorant transport to their olfactory epithelium ('olfactory flow'). Here we show that olfactory flow in the adult garpike Belone belone (Belonidae, Teleostei), a fish with an unusual nasal region, can be generated by its motion relative to water (swimming, or an external current, or both). We also show how the unusual features of the garpike's nasal region influence olfactory flow. These features comprise a triangular nasal cavity in which the olfactory epithelium is exposed to the external environment, a papilla situated within the nasal cavity, and an elongated ventral apex. To perform our investigation we first generated life-like plastic models of garpike heads from X-ray scans of preserved specimens. We then suspended these models in a flume and flowed water over them to simulate swimming. By directing filaments of dye at the static models, we were able to visualise flow in the nasal regions at physiologically relevant Reynolds numbers (700-2,000). We found that flow of water over the heads did cause circulation in the nasal cavity. Vortices may assist in this circulation. The pattern of olfactory flow was influenced by morphological variations and the asymmetry of the nasal region. The unusual features of the nasal region may improve odorant sampling in the garpike, by dispersing flow over the olfactory epithelium and by creating favourable conditions for odorant transport (e.g. steep velocity gradients). Unexpectedly, we found that the mouth and the base of the garpike's jaws may assist the sampling process. Thus, despite its apparent simplicity, the garpike's nasal region is likely to act as an effective trap for odorant molecules.

Exploring miniature insect brains using micro-CT scanning techniques.

The capacity to explore soft tissue structures in detail is important in understanding animal physiology and how this determines features such as movement, behaviour and the impact of trauma on regular function. Here we use advances in micro-computed tomography (micro-CT) technology to explore the brain of an important insect pollinator and model organism, the bumblebee (Bombus terrestris). Here we present a method for accurate imaging and exploration of insect brains that keeps brain tissue free from trauma and in its natural stereo-geometry, and showcase our 3D reconstructions and analyses of 19 individual brains at high resolution. Development of this protocol allows relatively rapid and cost effective brain reconstructions, making it an accessible methodology to the wider scientific community. The protocol describes the necessary steps for sample preparation, tissue staining, micro-CT scanning and 3D reconstruction, followed by a method for image analysis using the freeware SPIERS. These image analysis methods describe how to virtually extract key composite structures from the insect brain, and we demonstrate the application and precision of this method by calculating structural volumes and investigating the allometric relationships between bumblebee brain structures.

Evolutionary trade-off between vocal tract and testes dimensions in howler monkeys.

Males often face a trade-off between investments in precopulatory and postcopulatory traits [1], particularly when male-male contest competition determines access to mates [2]. To date, studies of precopulatory strategies have largely focused on visual ornaments (e.g., coloration) or weapon morphology (e.g., antlers, horns, and canines). However, vocalizations can also play an important role in both male competition and female choice [3-5]. We investigated variation in vocal tract dimensions among male howler monkeys (Alouatta spp.), which produce loud roars using a highly specialized and greatly enlarged hyoid bone and larynx [6]. We examined the relative male investment in hyoids and testes among howler monkey species in relation to the level of male-male competition and analyzed the acoustic consequences of variation in hyoid morphology. Species characterized by single-male groups have large hyoids and small testes, suggesting high levels of vocally mediated competition. Larger hyoids lower formant frequencies, probably increasing the acoustic impression of male body size and playing a role analogous to investment in large body size or weaponry. Across species, as the number of males per group increases, testes volume also increases, indicating higher levels of postcopulatory sperm competition, while hyoid volume decreases. These results provide the first evidence of an evolutionary trade-off between investment in precopulatory vocal characteristics and postcopulatory sperm production.

A Polychaete's powerful punch: venom gland transcriptomics of Glycera reveals a complex cocktail of toxin homologs.

Glycerids are marine annelids commonly known as bloodworms. Bloodworms have an eversible proboscis adorned with jaws connected to venom glands. Bloodworms prey on invertebrates, and it is known that the venom glands produce compounds that can induce toxic effects in animals. Yet, none of these putative toxins has been characterized on a molecular basis. Here we present the transcriptomic profiles of the venom glands of three species of bloodworm, Glycera dibranchiata, Glycera fallax and Glycera tridactyla, as well as the body tissue of G. tridactyla. The venom glands express a complex mixture of transcripts coding for putative toxin precursors. These transcripts represent 20 known toxin classes that have been convergently recruited into animal venoms, as well as transcripts potentially coding for Glycera-specific toxins. The toxins represent five functional categories: Pore-forming and membrane-disrupting toxins, neurotoxins, protease inhibitors, other enzymes, and CAP domain toxins. Many of the transcripts coding for putative Glycera toxins belong to classes that have been widely recruited into venoms, but some are homologs of toxins previously only known from the venoms of scorpaeniform fish and monotremes (stonustoxin-like toxin), turrid gastropods (turripeptide-like peptides), and sea anemones (gigantoxin I-like neurotoxin). This complex mixture of toxin homologs suggests that bloodworms employ venom while predating on macroscopic prey, casting doubt on the previously widespread opinion that G. dibranchiata is a detritivore. Our results further show that researchers should be aware that different assembly methods, as well as different methods of homology prediction, can influence the transcriptomic profiling of venom glands.