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Fluid preserved animal specimens in the collections of natural history museums constitute an invaluable archive of past and present animal diversity. Well-preserved specimens have a shelf-life spanning centuries and are widely used for e.g. anatomical, taxonomical and genetic studies. The way specimens were collected depended on the type of animal and the historical setting. As many small mammals and birds were historically collected by shooting, large quantities of heavy metal residues, primarily lead, may have been introduced into the sample in the form of lead shot pellets. Over time, these pellets may react with tissue fluids and/or the fixation and preservation agents and corrode into lead salts. As these chemicals are toxic, they could constitute a health issue to collection staff. Additionally, heavy element chemicals interfere with several imaging technologies increasingly used for non-invasive studies, and may confound anatomical and pathological investigations on affected specimens. Here we present a case-study based on platypus (Ornithorhynchus anatinus) and other small mammals containing lead pellets from the collection of The Natural History Museum of Denmark., Competing Interests: I have read the journal’s policy and the author Michiel Krols of this manuscript has the following competing interests: Employed at TESCAN XRE, a manufacture of one of the micro-CT systems used in the study (UniTOM XL Spectral). The remaining authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials, (Copyright: © 2024 Lauridsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The cardiac conduction system in large carnivores, such as the African lion (Panthera leo), represents a significant knowledge gap in both veterinary science and in cardiac electrophysiology. Short QT intervals have been reported from zoo-kept, anaesthetized lions, and our goal was to record the first ECGs from wild, conscious lions roaming freely, and compare them to zoo-kept lions under the hypothesis that short QT is unique to zoo-kept lions. Macroscopic and histological examinations were performed on heart tissue removed from nine healthy zoo lions. ECGs were recorded from the nine anaesthetized zoo-kept lions, and from 15 anaesthetized and conscious wild lions in Africa. Our histological and topographical description of the lion's heart matched what has previously been published. In conscious lions, the ECG recordings revealed a mean heart rate of 70 ± 4 beats/min, with faster heart rates during the night. PQ and QT intervals were heart rate dependent in the conscious lions. Interestingly, QT intervals recorded in wild lions were markedly longer than QT intervals from zoo lions (398 ± 40 vs. 297 ± 9 ms, respectively; P < 0.0001). Anaesthesia or heart rate did not account for this difference. We provide a comprehensive description of the cardiac anatomy and electrophysiology of wild and zoo-kept lions. QT intervals were significantly shorter in zoo lions, suggesting functional disparities in cardiac electrophysiology between wild and zoo-kept lions, potentially related to physical fitness. These findings underscore the plasticity of cardiac electrophysiology and may be of value when reintroducing endangered species into the wild and when managing lions in human care., (© 2024 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Despite tremendous efforts in the past decades, relationships among main avian lineages remain heavily debated without a clear resolution. Discrepancies have been attributed to diversity of species sampled, phylogenetic method and the choice of genomic regions 1-3 . Here we address these issues by analysing the genomes of 363 bird species 4 (218 taxonomic families, 92% of total). Using intergenic regions and coalescent methods, we present a well-supported tree but also a marked degree of discordance. The tree confirms that Neoaves experienced rapid radiation at or near the Cretaceous-Palaeogene boundary. Sufficient loci rather than extensive taxon sampling were more effective in resolving difficult nodes. Remaining recalcitrant nodes involve species that are a challenge to model due to either extreme DNA composition, variable substitution rates, incomplete lineage sorting or complex evolutionary events such as ancient hybridization. Assessment of the effects of different genomic partitions showed high heterogeneity across the genome. We discovered sharp increases in effective population size, substitution rates and relative brain size following the Cretaceous-Palaeogene extinction event, supporting the hypothesis that emerging ecological opportunities catalysed the diversification of modern birds. The resulting phylogenetic estimate offers fresh insights into the rapid radiation of modern birds and provides a taxon-rich backbone tree for future comparative studies., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Understanding the processes that determine how animals allocate time to space is a major challenge, although it is acknowledged that summed animal movement pathways over time must define space-time use. The critical question is then, what processes structure these pathways? Following the idea that turns within pathways might be based on environmentally determined decisions, we equipped Arabian oryx with head- and body-mounted tags to determine how they orientated their heads - which we posit is indicative of them assessing the environment - in relation to their movement paths, to investigate the role of environment scanning in path tortuosity. After simulating predators to verify that oryx look directly at objects of interest, we recorded that, during routine movement, > 60% of all turns in the animals' paths, before being executed, were preceded by a change in head heading that was not immediately mirrored by the body heading: The path turn angle (as indicated by the body heading) correlated with a prior change in head heading (with head heading being mirrored by subsequent turns in the path) twenty-one times more than when path turns occurred due to the animals adopting a body heading that went in the opposite direction to the change in head heading. Although we could not determine what the objects of interest were, and therefore the proposed reasons for turning, we suggest that this reflects the use of cephalic senses to detect advantageous environmental features (e.g. food) or to detect detrimental features (e.g. predators). The results of our pilot study suggest how turns might emerge in animal pathways and we propose that examination of points of inflection in highly resolved animal paths could represent decisions in landscapes and their examination could enhance our understanding of how animal pathways are structured., (© 2023. The Author(s).)

The germline mutation rate determines the pace of genome evolution and is an evolving parameter itself 1 . However, little is known about what determines its evolution, as most studies of mutation rates have focused on single species with different methodologies 2 . Here we quantify germline mutation rates across vertebrates by sequencing and comparing the high-coverage genomes of 151 parent-offspring trios from 68 species of mammals, fishes, birds and reptiles. We show that the per-generation mutation rate varies among species by a factor of 40, with mutation rates being higher for males than for females in mammals and birds, but not in reptiles and fishes. The generation time, age at maturity and species-level fecundity are the key life-history traits affecting this variation among species. Furthermore, species with higher long-term effective population sizes tend to have lower mutation rates per generation, providing support for the drift barrier hypothesis 3 . The exceptionally high yearly mutation rates of domesticated animals, which have been continually selected on fecundity traits including shorter generation times, further support the importance of generation time in the evolution of mutation rates. Overall, our comparative analysis of pedigree-based mutation rates provides ecological insights on the mutation rate evolution in vertebrates., (© 2023. The Author(s).)

Vocal learning is thought to have evolved in 3 orders of birds (songbirds, parrots, and hummingbirds), with each showing similar brain regions that have comparable gene expression specializations relative to the surrounding forebrain motor circuitry. Here, we searched for signatures of these same gene expression specializations in previously uncharacterized brains of 7 assumed vocal non-learning bird lineages across the early branches of the avian family tree. Our findings using a conserved marker for the song system found little evidence of specializations in these taxa, except for woodpeckers. Instead, woodpeckers possessed forebrain regions that were anatomically similar to the pallial song nuclei of vocal learning birds. Field studies of free-living downy woodpeckers revealed that these brain nuclei showed increased expression of immediate early genes (IEGs) when males produce their iconic drum displays, the elaborate bill-hammering behavior that individuals use to compete for territories, much like birdsong. However, these specialized areas did not show increased IEG expression with vocalization or flight. We further confirmed that other woodpecker species contain these brain nuclei, suggesting that these brain regions are a common feature of the woodpecker brain. We therefore hypothesize that ancient forebrain nuclei for refined motor control may have given rise to not only the song control systems of vocal learning birds, but also the drumming system of woodpeckers., Competing Interests: The authors have declared that no competing interests exist.

Comparative neuroimaging has been used to identify changes in white matter architecture across primate species phylogenetically close to humans, but few have compared the phylogenetically distant species. Here, we acquired postmortem diffusion imaging data from ring-tailed lemurs (Lemur catta), black-capped squirrel monkeys (Saimiri boliviensis), and rhesus macaques (Macaca mulatta). We were able to establish templates and surfaces allowing us to investigate sulcal, cortical, and white matter anatomy. The results demonstrate an expansion of the frontal projections of the superior longitudinal fasciculus complex in squirrel monkeys and rhesus macaques compared to ring-tailed lemurs, which correlates with sulcal anatomy and the lemur's smaller prefrontal granular cortex. The connectivity of the ventral pathway in the parietal region is also comparatively reduced in ring-tailed lemurs, with the posterior projections of the inferior longitudinal fasciculus not extending toward parietal cortical areas as in the other species. In the squirrel monkeys we note a very specific occipito-parietal anatomy that is apparent in their surface anatomy and the expansion of the posterior projections of the optical radiation. Our study supports the hypothesis that the connectivity of the prefrontal-parietal regions became relatively elaborated in the simian lineage after divergence from the prosimian lineage., (© The Author(s) 2021. Published by Oxford University Press.)

Background: The ring-tailed lemur (Lemur catta) is a charismatic strepsirrhine primate endemic to Madagascar. These lemurs are of particular interest, given their status as a flagship species and widespread publicity in the popular media. Unfortunately, a recent population decline has resulted in the census population decreasing to <2,500 individuals in the wild, and the species's classification as an endangered species by the IUCN. As is the case for most strepsirrhine primates, only a limited amount of genomic research has been conducted on L. catta, in part owing to the lack of genomic resources., Results: We generated a new high-quality reference genome assembly for L. catta (mLemCat1) that conforms to the standards of the Vertebrate Genomes Project. This new long-read assembly is composed of Pacific Biosciences continuous long reads (CLR data), Optical Mapping Bionano reads, Arima HiC data, and 10X linked reads. The contiguity and completeness of the assembly are extremely high, with scaffold and contig N50 values of 90.982 and 10.570 Mb, respectively. Additionally, when compared to other high-quality primate assemblies, L. catta has the lowest reported number of Alu elements, which results predominantly from a lack of AluS and AluY elements., Conclusions: mLemCat1 is an excellent genomic resource not only for the ring-tailed lemur community, but also for other members of the Lemuridae family, and is the first very long read assembly for a strepsirrhine., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Post-mortem magnetic resonance imaging (MRI) provides the opportunity to acquire high-resolution datasets to investigate neuroanatomy and validate the origins of image contrast through microscopy comparisons. We introduce the Digital Brain Bank (open.win.ox.ac.uk/DigitalBrainBank), a data release platform providing open access to curated, multimodal post-mortem neuroimaging datasets. Datasets span three themes -Digital Neuroanatomist : datasets for detailed neuroanatomical investigations; Digital Brain Zoo : datasets for comparative neuroanatomy; and Digital Pathologist : datasets for neuropathology investigations. The first Digital Brain Bank data release includes 21 distinctive whole-brain diffusion MRI datasets for structural connectivity investigations, alongside microscopy and complementary MRI modalities. This includes one of the highest-resolution whole-brain human diffusion MRI datasets ever acquired, whole-brain diffusion MRI in fourteen nonhuman primate species, and one of the largest post-mortem whole-brain cohort imaging studies in neurodegeneration. The Digital Brain Bank is the culmination of our lab's investment into post-mortem MRI methodology and MRI-microscopy analysis techniques. This manuscript provides a detailed overview of our work with post-mortem imaging to date, including the development of diffusion MRI methods to image large post-mortem samples, including whole, human brains. Taken together, the Digital Brain Bank provides cross-scale, cross-species datasets facilitating the incorporation of post-mortem data into neuroimaging studies., Competing Interests: BT, TH, OA, SB, GB, MB, KB, SF, Mv, AH, IH, AK, AL, PM, RM, JM, DM, MP, LR, JS, LS, CS, AS, MT, CW, RM No competing interests declared, SJ, KM Reviewing editor, eLife, (© 2022, Tendler et al.)

Biodiversity monitoring at the community scale is a critical element of assessing and studying species distributions, ecology, diversity, and movements, and it is key to understanding and tracking environmental and anthropogenic effects on natural ecosystems. 1-4 Vertebrates in terrestrial ecosystems are experiencing extinctions and declines in both population numbers and sizes due to increasing threats from human activities and environmental change. 5-8 Terrestrial vertebrate monitoring using existing methods is generally costly and laborious, and although environmental DNA (eDNA) is becoming the tool of choice to assess biodiversity, few sample types effectively capture terrestrial vertebrate diversity. We hypothesized that eDNA captured from air could allow straightforward collection and characterization of terrestrial vertebrate communities. We filtered air at three localities in the Copenhagen Zoo: a stable, outside between the outdoor enclosures, and in the Rainforest House. Through metabarcoding of airborne eDNA, we detected 49 vertebrate species spanning 26 orders and 37 families: 30 mammal, 13 bird, 4 fish, 1 amphibian, and 1 reptile species. These spanned animals kept at the zoo, species occurring in the zoo surroundings, and species used as feed in the zoo. The detected species comprise a range of taxonomic orders and families, sizes, behaviors, and abundances. We found shorter distance to the air sampling device and higher animal biomass to increase the probability of detection. We hereby show that airborne eDNA can offer a fundamentally new way of studying and monitoring terrestrial communities., Competing Interests: Declaration of interests M.S.J. is Chief Science Officer at AirLabs, the company that designed and built the 3D-printed housings for the particle samplers used in this study. The blueprints of these housings are freely available and provided in Figure S1. All other air sampling equipment, i.e., the water vacuum, blower fans used for the 24 V and 5 V particle samplers, and batteries, are available in commercial companies not related to Airlabs. Thereby, the current study is not of direct commercial value to Airlabs. The remaining authors declare that they have no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The combined use of global positioning system (GPS) technology and motion sensors within the discipline of movement ecology has increased over recent years. This is particularly the case for instrumented wildlife, with many studies now opting to record parameters at high (infra-second) sampling frequencies. However, the detail with which GPS loggers can elucidate fine-scale movement depends on the precision and accuracy of fixes, with accuracy being affected by signal reception. We hypothesized that animal behaviour was the main factor affecting fix inaccuracy, with inherent GPS positional noise (jitter) being most apparent during GPS fixes for non-moving locations, thereby producing disproportionate error during rest periods. A movement-verified filtering (MVF) protocol was constructed to compare GPS-derived speed data with dynamic body acceleration, to provide a computationally quick method for identifying genuine travelling movement. This method was tested on 11 free-ranging lions ( Panthera leo ) fitted with collar-mounted GPS units and tri-axial motion sensors recording at 1 and 40 Hz, respectively. The findings support the hypothesis and show that distance moved estimates were, on average, overestimated by greater than 80% prior to GPS screening. We present the conceptual and mathematical protocols for screening fix inaccuracy within high-resolution GPS datasets and demonstrate the importance that MVF has for avoiding inaccurate and biased estimates of movement.

Background: Understanding what animals do in time and space is important for a range of ecological questions, however accurate estimates of how animals use space is challenging. Within the use of animal-attached tags, radio telemetry (including the Global Positioning System, 'GPS') is typically used to verify an animal's location periodically. Straight lines are typically drawn between these 'Verified Positions' ('VPs') so the interpolation of space-use is limited by the temporal and spatial resolution of the system's measurement. As such, parameters such as route-taken and distance travelled can be poorly represented when using VP systems alone. Dead-reckoning has been suggested as a technique to improve the accuracy and resolution of reconstructed movement paths, whilst maximising battery life of VP systems. This typically involves deriving travel vectors from motion sensor systems and periodically correcting path dimensions for drift with simultaneously deployed VP systems. How often paths should be corrected for drift, however, has remained unclear., Methods and Results: Here, we review the utility of dead-reckoning across four contrasting model species using different forms of locomotion (the African lion Panthera leo , the red-tailed tropicbird Phaethon rubricauda , the Magellanic penguin Spheniscus magellanicus , and the imperial cormorant Leucocarbo atriceps ). Simulations were performed to examine the extent of dead-reckoning error, relative to VPs, as a function of Verified Position correction (VP correction) rate and the effect of this on estimates of distance moved. Dead-reckoning error was greatest for animals travelling within air and water. We demonstrate how sources of measurement error can arise within VP-corrected dead-reckoned tracks and propose advancements to this procedure to maximise dead-reckoning accuracy., Conclusions: We review the utility of VP-corrected dead-reckoning according to movement type and consider a range of ecological questions that would benefit from dead-reckoning, primarily concerning animal-barrier interactions and foraging strategies., Competing Interests: Competing interests The authors declare no conflict of interest.

Elephant endotheliotropic herpesvirus haemorrhagic disease (EEHV-HD) is widely acknowledged as the most common cause of mortality in young Asian elephants (Elephas maximus) in captivity. The objective of the current study was to perform a blinded, retrospective pathology review of European EEHV-HD fatalities, constituting the largest systematic assessment of EEHV-HD pathology to date. Findings between viral genotypes were compared with the aim to investigate if disseminated intravascular coagulation (DIC) could be substantiated as a significant complicating factor, thereby increasing the understanding of disease pathophysiology. Immunohistochemical staining confirmed endothelial cell (EC) damage and the presence of EC intranuclear inclusion bodies, demonstrating a direct viral cytopathic effect. Microthrombi were observed in 63% of cases in several organs, including lungs, which, together with widespread haemorrhage and thrombocytopenia reported in EEHV-HD case reports, supports the presence of overt DIC as a serious haemostatic complication of active EEHV infection. Death was attributed to widespread vascular damage with multi-organ dysfunction, including severe acute myocardial haemorrhage and subsequent cardiac failure. Systemic inflammation observed in the absence of bacterial infection may be caused by cytokine release syndrome. Findings reinforce the necessity to investigate cytokine responses and haemostatic status during symptomatic and asymptomatic EEHV viraemia, to potentially support the use of anti-inflammatory treatment in conjunction with anti-viral therapy and cardiovascular support.

The evolved adaptations of other species can be a source of insight for novel biomedical innovation. Limitations of traditional animal models for the study of some pathologies are fueling efforts to find new approaches to biomedical investigation. One emerging approach recognizes the evolved adaptations in other species as possible solutions to human pathology. The giraffe heart, for example, appears resistant to pathology related to heart failure with preserved ejection fraction (HFpEF)-a leading form of hypertension-associated cardiovascular disease in humans. Here, we postulate that the physiological pressure-induced left ventricular thickening in giraffes does not result in the pathological cardiovascular changes observed in humans with hypertension. The mechanisms underlying this cardiovascular adaptation to high blood pressure in the giraffe may be a bioinspired roadmap for preventive and therapeutic strategies for human HFpEF., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health.)

To elucidate factors underlying the evolution of large brains in cetaceans, we examined 16 brains from 14 cetartiodactyl species, with immunohistochemical techniques, for evidence of non-shivering thermogenesis. We show that, in comparison to the 11 artiodactyl brains studied (from 11 species), the 5 cetacean brains (from 3 species), exhibit an expanded expression of uncoupling protein 1 (UCP1, UCPs being mitochondrial inner membrane proteins that dissipate the proton gradient to generate heat) in cortical neurons, immunolocalization of UCP4 within a substantial proportion of glia throughout the brain, and an increased density of noradrenergic axonal boutons (noradrenaline functioning to control concentrations of and activate UCPs). Thus, cetacean brains studied possess multiple characteristics indicative of intensified thermogenetic functionality that can be related to their current and historical obligatory aquatic niche. These findings necessitate reassessment of our concepts regarding the reasons for large brain evolution and associated functional capacities in cetaceans.

Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity 1-4 . Sparse taxon sampling has previously been proposed to confound phylogenetic inference 5 , and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families-including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species.

The use of inhalational anaesthesia is ubiquitous in terrestrial vertebrates. Given the dependence of these agents on delivery by the cardiorespiratory system, we developed a new computational model predicting equilibration of inhaled anaesthetics in mammalian and ectotherm conditions including the ability of reptiles to maintain vascular shunts. A multi-compartment model was constructed from simultaneously-solved equations, verified by comparison to the literature for endo and ectotherm physiology. The time to 90% equilibration of anaesthetic in arterial blood (t 90 ) is predicted and used to compare anaesthetics and physiologies. The five to tenfold lower cardiac output and minute ventilation of ectothermic vertebrates is predicted to slow equilibration times by five to ten times leading to 90% equilibration in ectotherm arterial blood of over 200 min, compounded by reduction in body temperature, and the extent of right-to-left vascular shunts. The impact of these findings is also influenced by the solubility coefficient of the anaesthetic, such that at net right-to-left shunt fractions of over 0.8, sevoflurane loses the advantage of faster equilibration, in comparison with isoflurane. We explore clinical strategies to regulate anaesthetic uptake in ectotherms by managing convectional flow especially by supportive ventilation and reduction of the right-to-left shunt.

Background: The hamadryas baboon (Papio hamadryas) is a highly social primate that lives in complex multilevel societies exhibiting a wide range of group behaviors akin to humans. In contrast to the widely studied human microbiome, there is a paucity of information on the host-associated microbiomes of nonhuman primates (NHPs). Here, our goal was to understand the microbial composition throughout different body sites of cohabiting baboons., Results: We analyzed 170 oral, oropharyngeal, cervical, uterine, vaginal, nasal and rectal samples from 16 hamadryas baboons via 16S rRNA gene sequencing. Additionally, raw Miseq sequencing data from 1041 comparable publicly available samples from the human oral cavity, gut and vagina were reanalyzed using the same pipeline. We compared the baboon and human microbiome of the oral cavity, gut and vagina, showing that the baboon microbiome is distinct from the human. Baboon cohabitants share similar microbial profiles in their cervix, uterus, vagina, and gut. The oral cavity, gut and vagina shared more bacterial amplicon sequence variants (ASVs) in group living baboons than in humans. The shared ASVs had significantly positive correlations between most body sites, suggesting a potential bacterial exchange throughout the body. No significant differences in gut microbiome composition were detected within the maternity line and between maternity lines, suggesting that the offspring gut microbiota is shaped primarily through bacterial exchange among cohabitants. Finally, Lactobacillus was not so predominant in baboon vagina as in the human vagina but was the most abundant genus in the baboon gut., Conclusions: This study is the first to provide comprehensive analyses of the baboon microbiota across different body sites. We contrast this to human body sites and find substantially different microbiomes. This group of cohabitating baboons generally showed higher microbial diversity and remarkable similarities between body sites than were observed in humans. These data and findings from one group of baboons can form the basis of future microbiome studies in baboons and be used as a reference in research where the microbiome is expected to impact human modeling with baboons.

Background: Videographic material of animals can contain inapparent signals, such as color changes or motion that hold information about physiological functions, such as heart and respiration rate, pulse wave velocity, and vocalization. Eulerian video magnification allows the enhancement of such signals to enable their detection. The purpose of this study is to demonstrate how signals relevant to experimental physiology can be extracted from non-contact videographic material of animals., Results: We applied Eulerian video magnification to detect physiological signals in a range of experimental models and in captive and free ranging wildlife. Neotenic Mexican axolotls were studied to demonstrate the extraction of heart rate signal of non-embryonic animals from dedicated videographic material. Heart rate could be acquired both in single and multiple animal setups of leucistic and normally colored animals under different physiological conditions (resting, exercised, or anesthetized) using a wide range of video qualities. Pulse wave velocity could also be measured in the low blood pressure system of the axolotl as well as in the high-pressure system of the human being. Heart rate extraction was also possible from videos of conscious, unconstrained zebrafish and from non-dedicated videographic material of sand lizard and giraffe. This technique also allowed for heart rate detection in embryonic chickens in ovo through the eggshell and in embryonic mice in utero and could be used as a gating signal to acquire two-phase volumetric micro-CT data of the beating embryonic chicken heart. Additionally, Eulerian video magnification was used to demonstrate how vocalization-induced vibrations can be detected in infrasound-producing Asian elephants., Conclusions: Eulerian video magnification provides a technique to extract inapparent temporal signals from videographic material of animals. This can be applied in experimental and comparative physiology where contact-based recordings (e.g., heart rate) cannot be acquired.

The ejection fraction of the trabeculated cardiac ventricle of reptiles has not previously been measured. Here, we used the gold standard clinical methodology - electrocardiogram-gated flow magnetic resonance imaging (MRI) - to validate stroke volume measurements and end diastolic ventricular blood volume. This produced an estimate of ejection fraction in our study species, the red footed tortoise Chelonoidis carbonarius ( n =5), under isoflurane anaesthesia of 88±11%. After reduction of the prevailing right-to-left intraventricular shunt through the action of atropine, the ejection fraction was 96±6%. This methodology opens new avenues for studying the complex hearts of ectotherms, and validating hypotheses on the function of a more highly trabeculated heart than that of endotherms, which have lower ejection fractions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: Penguins (Sphenisciformes) are a remarkable order of flightless wing-propelled diving seabirds distributed widely across the southern hemisphere. They share a volant common ancestor with Procellariiformes close to the Cretaceous-Paleogene boundary (66 million years ago) and subsequently lost the ability to fly but enhanced their diving capabilities. With ∼20 species among 6 genera, penguins range from the tropical Galápagos Islands to the oceanic temperate forests of New Zealand, the rocky coastlines of the sub-Antarctic islands, and the sea ice around Antarctica. To inhabit such diverse and extreme environments, penguins evolved many physiological and morphological adaptations. However, they are also highly sensitive to climate change. Therefore, penguins provide an exciting target system for understanding the evolutionary processes of speciation, adaptation, and demography. Genomic data are an emerging resource for addressing questions about such processes., Results: Here we present a novel dataset of 19 high-coverage genomes that, together with 2 previously published genomes, encompass all extant penguin species. We also present a well-supported phylogeny to clarify the relationships among penguins. In contrast to recent studies, our results demonstrate that the genus Aptenodytes is basal and sister to all other extant penguin genera, providing intriguing new insights into the adaptation of penguins to Antarctica. As such, our dataset provides a novel resource for understanding the evolutionary history of penguins as a clade, as well as the fine-scale relationships of individual penguin lineages. Against this background, we introduce a major consortium of international scientists dedicated to studying these genomes. Moreover, we highlight emerging issues regarding ensuring legal and respectful indigenous consultation, particularly for genomic data originating from New Zealand Taonga species., Conclusions: We believe that our dataset and project will be important for understanding evolution, increasing cultural heritage and guiding the conservation of this iconic southern hemisphere species assemblage., (© The Author(s) 2019. Published by Oxford University Press.)

Lead (Pb) exposure is associated with adverse health effects in both humans and wildlife. Blood lead levels (BLL) of sentinel wildlife species can be used to monitor environmental lead exposure and ecosystem health. BLL analyzers, such as the LeadCare®, are validated for use in humans, assessed for use in some avian species and cattle, and are increasingly being used on wildlife to monitor lead exposure. The LeadCare® analyzers use a technique called anodic stripping voltammetry (ASV). Species-specific conversion equations have been proposed to approximate the levels found with gold standard measuring methods such as inductively coupled plasma mass spectrometry (ICP-MS) because the ASV method has been shown to underestimate BLL in some species. In this study we assessed the LeadCare® Plus (LCP) for use on Scandinavian brown bears ( Ursus arctos ). LCP measurements were correlated with ICP-MS with a Bland-Altman analyzed bias of 16.3-22.5%, showing a consistent overestimation of BLL analyzed with LCP. Based on this analysis we provide conversion equations for calculating ICP-MS BLL based on the LCP results in Scandinavian brown bears. Our study shows that the LeadCare® Plus can be used for monitoring of lead exposure by approximating gold standard levels using conversion equations. This enables comparison with other gold standard measured BLL within the observed range of this study (38.20-174.00 μg/L). Our study also found that Scandinavian brown bears are highly exposed to environmental lead.

Because the white matter of the cerebral cortex contains axons that connect distant neurons in the cortical gray matter, the relationship between the volumes of the 2 cortical compartments is key for information transmission in the brain. It has been suggested that the volume of the white matter scales universally as a function of the volume of the gray matter across mammalian species, as would be expected if a global principle of wiring minimization applied. Using a systematic analysis across several mammalian clades, here we show that the volume of the white matter does not scale universally with the volume of the gray matter across mammals and is not optimized for wiring minimization. Instead, the ratio between volumes of gray and white matter is universally predicted by the same equation that predicts the degree of folding of the cerebral cortex, given the clade-specific scaling of cortical thickness, such that the volume of the gray matter (or the ratio of gray to total cortical volumes) divided by the square root of cortical thickness is a universal function of total cortical volume, regardless of the number of cortical neurons. Thus, the very mechanism that we propose to generate cortical folding also results in compactness of the white matter to a predictable degree across a wide variety of mammalian species., Competing Interests: The authors declare no conflict of interest.

Inhalant anesthesia is challenging in chelonians due to a great capacity for breath-holding and an incomplete separation of the cardiac ventricle. Deoxygenated blood can recirculate back into systemic circulation by bypassing the lung in a process referred to as intracardiac right to left (R-L) shunting. Via electrocardiogram gated magnetic resonance imaging, a novel modality to investigate arterial flows in reptiles, intracardiac shunting and its elimination via atropine during gas anesthesia in tortoises (Chelonoidis carbonaria) was demonstrated. The great vessels of the heart were visualized confirming that after shunt-elimination, the flow (mean ± sd) in the pulmonary arteries increased significantly (54.6 ± 9.5 mL min -1 kg -1 vs 10.8 ± 3.4 mL min -1 kg -1 ; P < 0.008). Consequently, animals required significantly lower concentrations of inhaled anesthetics to maintain a stable anesthesia. To that end, the minimum anesthetic concentration (MAC) of isoflurane needed to maintain surgical anesthesia was measured. A significantly lower MAC was found after administration of atropine (mean MAC ± sd 2.2 ± 0.3% vs 3.2 ± 0.4%; P < 0.002). Previously, MAC has been indeterminable in chelonians likely due to intracardiac shunting, so this report constitutes the first MAC study performed in a tortoise.

Donkeys and horses share a common ancestor dating back to about 4 million years ago. Although a high-quality genome assembly at the chromosomal level is available for the horse, current assemblies available for the donkey are limited to moderately sized scaffolds. The absence of a better-quality assembly for the donkey has hampered studies involving the characterization of patterns of genetic variation at the genome-wide scale. These range from the application of genomic tools to selective breeding and conservation to the more fundamental characterization of the genomic loci underlying speciation and domestication. We present a new high-quality donkey genome assembly obtained using the Chicago HiRise assembly technology, providing scaffolds of subchromosomal size. We make use of this new assembly to obtain more accurate measures of heterozygosity for equine species other than the horse, both genome-wide and locally, and to detect runs of homozygosity potentially pertaining to positive selection in domestic donkeys. Finally, this new assembly allowed us to identify fine-scale chromosomal rearrangements between the horse and the donkey that likely played an active role in their divergence and, ultimately, speciation.

Carnivorans are a diverse group of mammals that includes carnivorous, omnivorous and herbivorous, domesticated and wild species, with a large range of brain sizes. Carnivory is one of several factors expected to be cognitively demanding for carnivorans due to a requirement to outsmart larger prey. On the other hand, large carnivoran species have high hunting costs and unreliable feeding patterns, which, given the high metabolic cost of brain neurons, might put them at risk of metabolic constraints regarding how many brain neurons they can afford, especially in the cerebral cortex. For a given cortical size, do carnivoran species have more cortical neurons than the herbivorous species they prey upon? We find they do not; carnivorans (cat, mongoose, dog, hyena, lion) share with non-primates, including artiodactyls (the typical prey of large carnivorans), roughly the same relationship between cortical mass and number of neurons, which suggests that carnivorans are subject to the same evolutionary scaling rules as other non-primate clades. However, there are a few important exceptions. Carnivorans stand out in that the usual relationship between larger body, larger cortical mass and larger number of cortical neurons only applies to small and medium-sized species, and not beyond dogs: we find that the golden retriever dog has more cortical neurons than the striped hyena, African lion and even brown bear, even though the latter species have up to three times larger cortices than dogs. Remarkably, the brown bear cerebral cortex, the largest examined, only has as many neurons as the ten times smaller cat cerebral cortex, although it does have the expected ten times as many non-neuronal cells in the cerebral cortex compared to the cat. We also find that raccoons have dog-like numbers of neurons in their cat-sized brain, which makes them comparable to primates in neuronal density. Comparison of domestic and wild species suggests that the neuronal composition of carnivoran brains is not affected by domestication. Instead, large carnivorans appear to be particularly vulnerable to metabolic constraints that impose a trade-off between body size and number of cortical neurons.

Heterothermy, the ability to allow body temperature (Tb) to fluctuate, has been proposed as an adaptive mechanism that enables large ungulates to cope with the high environmental temperatures and lack of free water experienced in arid environments. By storing heat during the daytime and dissipating it during the night, arid-adapted ungulates may reduce evaporative water loss and conserve water. Adaptive heterothermy in large ungulates should be particularly pronounced in hot environments with severely limited access to free water. In the current study we investigated the effects of environmental temperature (ambient, Ta and soil, Ts) and water stress on the Tb of wild, free-ranging Arabian oryx (Oryx leucoryx) in two different sites in Saudi Arabia, Mahazat as-Sayd (MS) and Uruq Bani Ma'arid (UBM). Using implanted data loggers wet took continuous Tb readings every 10 minutes for an entire calendar year and determined the Tb amplitude as well as the heterothermy index (HI). Both differed significantly between sites but contrary to our expectations they were greater in MS despite its lower environmental temperatures and higher rainfall. This may be partially attributable to a higher activity in an unfamiliar environment for translocated animals in UBM. As expected Tb amplitude and HI were greatest during summer. Only minor sex differences were apparent that may be attributable to sex-specific investment into reproduction (e.g. male-male competition) during rut. Our results suggest that the degree of heterothermy is not only driven by extrinsic factors (e.g. environmental temperatures and water availability), but may also be affected by intrinsic factors (e.g. sex and/or behaviour).

Background: Covering the eye of all snakes is a transparent integumental structure known as the spectacle. In order to determine variations in spectacle thickness among species, the spectacles of 217 alcohol-preserved museum specimens of 44 species belonging to 14 different families underwent optical coherence tomography (OCT) to measure spectacular thickness. Multivariable analyses were made to determine whether family, activity period (diurnal/nocturnal) and habitat (arboreal/terrestrial/fossorial/aquatic) influenced spectacle thickness., Results: The thinnest spectacles in absolute terms were found in the Usambara bush viper (Viperidae) with a thickness of 74 ± 9 μm and the absolute thickest spectacle was found in the red-tailed pipe snake (Cylindrophiidae) which had a spectacle thickness of 244 ± 57 μm. Fossorial and aquatic snakes had significantly thicker spectacles than arboreal and terrestrial snakes. When spectacle thickness was correlated to eye size (horizontal spectacle diameter), Gray's earth snake (Uropeltidae) had the lowest ratio (1:7) and the cottonmouth (Viperidae) had the highest ratio (1:65). Multivariable and phylogenetic analyses showed that spectacular thickness could be predicted by taxonomic family and habitat, but not activity period., Conclusion: This phylogenetically broad systematic study of the thickness of the snake spectacle showed that spectacular thickness varies greatly across snake species and may reflect evolutionary adaptation and development.

The Chelonid herpesvirus 5 (ChHV5) has been consistently associated with fibropapillomatosis (FP), a transmissible neoplastic disease of marine turtles. Whether ChHV5 plays a causal role remains debated, partly because while FP tumours have been clearly documented to contain high concentrations of ChHV5 DNA, recent PCR-based studies have demonstrated that large proportions of asymptomatic marine turtles are also carriers of ChHV5. We used a real-time PCR assay to quantify the levels of ChHV5 Glycoprotein B (gB) DNA in both tumour and non-tumour skin tissues, from clinically affected and healthy turtles drawn from distant ocean basins across four species. In agreement with previous studies, higher ratios of viral to host DNA were consistently observed in tumour versus non-tumour tissues in turtles with FP. Unexpectedly however, the levels of ChHV5 gB DNA in clinically healthy turtles were significantly higher than in non-tumour tissues from FP positive turtles. Thus, a large proportion of clinically healthy sea turtle populations worldwide across species carry ChHV5 gB DNA presumably through persistent latent infections. ChHV5 appears to be ubiquitous regardless of the animals' clinical conditions. Hence, these results support the theory that ChHV5 is a near ubiquitous virus with latency characteristics requiring co-factors, possibly environmental or immune related, to induce FP.

Giraffes--the tallest extant animals on Earth--are renowned for their high central arterial blood pressure, which is necessary to secure brain perfusion. Arterial pressure may exceed 300 mmHg and has historically been attributed to an exceptionally large heart. Recently, this has been refuted by several studies demonstrating that the mass of giraffe heart is similar to that of other mammals when expressed relative to body mass. It thus remains unexplained how the normal-sized giraffe heart generates such massive arterial pressures. We hypothesized that giraffe hearts have a small intraventricular cavity and a relatively thick ventricular wall, allowing for generation of high arterial pressures at normal left ventricular wall tension. In nine anaesthetized giraffes (495±38 kg), we determined in vivo ventricular dimensions using echocardiography along with intraventricular and aortic pressures to calculate left ventricular wall stress. Cardiac output was also determined by inert gas rebreathing to provide an additional and independent estimate of stroke volume. Echocardiography and inert gas-rebreathing yielded similar cardiac outputs of 16.1±2.5 and 16.4±1.4 l min(-1), respectively. End-diastolic and end-systolic volumes were 521±61 ml and 228±42 ml, respectively, yielding an ejection fraction of 56±4% and a stroke volume of 0.59 ml kg(-1). Left ventricular circumferential wall stress was 7.83±1.76 kPa. We conclude that, relative to body mass, a small left ventricular cavity and a low stroke volume characterizes the giraffe heart. The adaptations result in typical mammalian left ventricular wall tensions, but produce a lowered cardiac output., (© 2016. Published by The Company of Biologists Ltd.)

Background: Trichuris suis and T. trichiura are two different whipworm species that infect pigs and humans, respectively. T. suis is found in pigs worldwide while T. trichiura is responsible for nearly 460 million infections in people, mainly in areas of poor sanitation in tropical and subtropical areas. The evolutionary relationship and the historical factors responsible for this worldwide distribution are poorly understood. In this study, we aimed to reconstruct the demographic history of Trichuris in humans and pigs, the evolutionary origin of Trichuris in these hosts and factors responsible for parasite dispersal globally., Methods: Parts of the mitochondrial nad1 and rrnL genes were sequenced followed by population genetic and phylogenetic analyses. Populations of Trichuris examined were recovered from humans (n = 31), pigs (n = 58) and non-human primates (n = 49) in different countries on different continents, namely Denmark, USA, Uganda, Ecuador, China and St. Kitts (Caribbean). Additional sequences available from GenBank were incorporated into the analyses., Results: We found no differentiation between human-derived Trichuris in Uganda and the majority of the Trichuris samples from non-human primates suggesting a common African origin of the parasite, which then was transmitted to Asia and further to South America. On the other hand, there was no differentiation between pig-derived Trichuris from Europe and the New World suggesting dispersal relates to human activities by transporting pigs and their parasites through colonisation and trade. Evidence for recent pig transport from China to Ecuador and from Europe to Uganda was also observed from their parasites. In contrast, there was high genetic differentiation between the pig Trichuris in Denmark and China in concordance with the host genetics., Conclusions: We found evidence for an African origin of T. trichiura which were then transmitted with human ancestors to Asia and further to South America. A host shift to pigs may have occurred in Asia from where T. suis seems to have been transmitted globally by a combination of natural host dispersal and anthropogenic factors.

Background: Free-ranging brown bears exhibit highly contrasting physiological states throughout the year. They hibernate 6 months of the year, experiencing a decrease in body temperature, heart rate, respiratory rate and metabolism. An increase in food consumption and the resulting weight gain (mostly through fat storage) prior to hibernation are also part of the brown bear's annual cycle. Due to these physiological changes, haematological and biochemical variables vary dramatically throughout the year. Seasonal changes in 12 haematological and 34 biochemical variables were evaluated in blood samples collected from 40 free-ranging subadult brown bears (22 females, 18 males) immobilised in Sweden in winter (February-March), spring (April-May), and summer (June)., Results: Higher levels of haemoglobin, haematocrit and red blood cell count, and a lower white blood cell count and mean cell volume was found during hibernation than in spring and summer. Lower values of the enzymes; aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), γ-glutamyl transpeptidase (GGT), glutamate dehydrogenase (GD) and amylase, and increased values of β-hydroxybutyrate (β-HBA) and blood lipids; triglycerides, cholesterol and free fatty acids, were present during hibernation compared to spring and summer., Conclusions: This study documents significant shifts in haematological and biochemical variables in samples collected from brown bears anaesthetised in winter (February-March) compared to in spring and summer (April-June), reflecting the lowered metabolic, renal and hepatic activity during hibernation. Lower values of enzymes and higher values of blood lipids during hibernation, likely reflect a lipid-based metabolism.

To investigate aquatic bird bornavirus 1 in Europe, we examined 333 brains from hunter-killed geese in Denmark in 2014. Seven samples were positive by reverse transcription PCR and were 98.2%-99.8% identical; they were also 97.4%-98.1% identical to reference strains of aquatic bird bornavirus 1 from geese in North America.

The histologic features of abnormal spectacles in 60 snakes from the 5 families of Boidae, Colubridae, Elapidae, Pythonidae, and Viperidae are described in a retrospective study conducted on specimens submitted to a private diagnostic service during a period of 15 years. Fifty-two snakes had inflammatory reactions in the spectacle. The stroma and outer epithelium of the spectacle were the layers most often involved in inflammatory disease. Lesions of the outer epithelium included edema, hyperkeratosis, and granulocyte infiltration occasionally with bacterial colonies and fungal elements. The stroma had infectious agents and inflammatory reactions in vessels and between the collagen fibrils. The inner epithelium had varying degrees of hyperplasia and hypertrophy, but no infectious agents were seen. Infectious agents in these cases included mites, bacterial disease, fungal disease, or a combination of bacterial and fungal disease. Special stains identified the bacteria most commonly involved to be Gram-positive cocci. Thirteen snakes had dysecdysis of the spectacle. Of these, 5 displayed a concurrent inflammatory reaction of the spectacle, while the remaining 8 snakes had extra keratin layers on a spectacle with an otherwise normal appearance. These keratin layers were attached to serocellular crusts located on the inner surface of the periocular scales. The cause for dyskeratotic lesions of the spectacle was not always apparent, and concurrent acariasis, other forms of dermatitis, trauma, suboptimal husbandry, and visceral disease were considered possible contributing factors. It was notable that only 4% of the submitted cases were found to have spectaculitis and/or spectacular dysecdysis., (© The Author(s) 2015.)

Ruminantia, the ruminating, hoofed mammals (cow, deer, giraffe and allies) are an unranked artiodactylan clade. Around 50-60 million years ago the BovB retrotransposon entered the ancestral ruminantian genome through horizontal gene transfer. A survey genome screen using 454-pyrosequencing of the Java mouse deer (Tragulus javanicus) and the lesser kudu (Tragelaphus imberbis) was done to investigate and to compare the landscape of transposable elements within Ruminantia. The family Tragulidae (mouse deer) is the only representative of Tragulina and phylogenetically important, because it represents the earliest divergence in Ruminantia. The data analyses show that, relative to other ruminantian species, the lesser kudu genome has seen an expansion of BovB Long INterspersed Elements (LINEs) and BovB related Short INterspersed Elements (SINEs) like BOVA2. In comparison the genome of Java mouse deer has fewer BovB elements than other ruminants, especially Bovinae, and has in addition a novel CHR-3 SINE most likely propagated by LINE-1. By contrast the other ruminants have low amounts of CHR SINEs but high numbers of actively propagating BovB-derived and BovB-propagated SINEs. The survey sequencing data suggest that the transposable element landscape in mouse deer (Tragulina) is unique among Ruminantia, suggesting a lineage specific evolutionary trajectory that does not involve BovB mediated retrotransposition. This shows that the genomic landscape of mobile genetic elements can rapidly change in any lineage., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recent studies have reported multiple cases of molecular adaptation in cetaceans related to their aquatic abilities. However, none of these has included the hippopotamus, precluding an understanding of whether molecular adaptations in cetaceans occurred before or after they split from their semi-aquatic sister taxa. Here, we obtained new transcriptomes from the hippopotamus and humpback whale, and analysed these together with available data from eight other cetaceans. We identified more than 11 000 orthologous genes and compiled a genome-wide dataset of 6845 coding DNA sequences among 23 mammals, to our knowledge the largest phylogenomic dataset to date for cetaceans. We found positive selection in nine genes on the branch leading to the common ancestor of hippopotamus and whales, and 461 genes in cetaceans compared to 64 in hippopotamus. Functional annotation revealed adaptations in diverse processes, including lipid metabolism, hypoxia, muscle and brain function. By combining these findings with data on protein-protein interactions, we found evidence suggesting clustering among gene products relating to nervous and muscular systems in cetaceans. We found little support for shared ancestral adaptations in the two taxa; most molecular adaptations in extant cetaceans occurred after their split with hippopotamids.

Background: Hemostasis is a defense mechanism that enhances an organism's survival by minimizing blood loss upon vascular injury. In vertebrates, hemostasis has been evolving with the cardio-vascular and hemodynamic systems over the last 450 million years. Birds and mammals have very similar vascular and hemodynamic systems, thus the mechanism that blocks ruptures in the vasculature is expected to be the same. However, the speed of the process varies across vertebrates, and is particularly slow for birds. Understanding the differences in the hemostasis pathway between birds and mammals, and placing them in perspective to other vertebrates may provide clues to the genetic contribution to variation in blood clotting phenotype in vertebrates. We compiled genomic data corresponding to key elements involved in hemostasis across vertebrates to investigate its genetic basis and understand how it affects fitness., Results: We found that: i) fewer genes are involved in hemostasis in birds compared to mammals; and ii) the largest differences concern platelet membrane receptors and components from the kallikrein-kinin system. We propose that lack of the cytoplasmic domain of the GPIb receptor subunit alpha could be a strong contributor to the prolonged bleeding phenotype in birds. Combined analysis of laboratory assessments of avian hemostasis with the first avian phylogeny based on genomic-scale data revealed that differences in hemostasis within birds are not explained by phylogenetic relationships, but more so by genetic variation underlying components of the hemostatic process, suggestive of natural selection., Conclusions: This work adds to our understanding of the evolution of hemostasis in vertebrates. The overlap with the inflammation, complement and renin-angiotensin (blood pressure regulation) pathways is a potential driver of rapid molecular evolution in the hemostasis network. Comparisons between avian species and mammals allowed us to hypothesize that the observed mammalian innovations might have contributed to the diversification of mammals that give birth to live young.

The ability to imitate complex sounds is rare, and among birds has been found only in parrots, songbirds, and hummingbirds. Parrots exhibit the most advanced vocal mimicry among non-human animals. A few studies have noted differences in connectivity, brain position and shape in the vocal learning systems of parrots relative to songbirds and hummingbirds. However, only one parrot species, the budgerigar, has been examined and no differences in the presence of song system structures were found with other avian vocal learners. Motivated by questions of whether there are important differences in the vocal systems of parrots relative to other vocal learners, we used specialized constitutive gene expression, singing-driven gene expression, and neural connectivity tracing experiments to further characterize the song system of budgerigars and/or other parrots. We found that the parrot brain uniquely contains a song system within a song system. The parrot "core" song system is similar to the song systems of songbirds and hummingbirds, whereas the "shell" song system is unique to parrots. The core with only rudimentary shell regions were found in the New Zealand kea, representing one of the only living species at a basal divergence with all other parrots, implying that parrots evolved vocal learning systems at least 29 million years ago. Relative size differences in the core and shell regions occur among species, which we suggest could be related to species differences in vocal and cognitive abilities.

The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Horses, asses, and zebras belong to a single genus, Equus, which emerged 4.0-4.5 Mya. Although the equine fossil record represents a textbook example of evolution, the succession of events that gave rise to the diversity of species existing today remains unclear. Here we present six genomes from each living species of asses and zebras. This completes the set of genomes available for all extant species in the genus, which was hitherto represented only by the horse and the domestic donkey. In addition, we used a museum specimen to characterize the genome of the quagga zebra, which was driven to extinction in the early 1900s. We scan the genomes for lineage-specific adaptations and identify 48 genes that have evolved under positive selection and are involved in olfaction, immune response, development, locomotion, and behavior. Our extensive genome dataset reveals a highly dynamic demographic history with synchronous expansions and collapses on different continents during the last 400 ky after major climatic events. We show that the earliest speciation occurred with gene flow in Northern America, and that the ancestor of present-day asses and zebras dispersed into the Old World 2.1-3.4 Mya. Strikingly, we also find evidence for gene flow involving three contemporary equine species despite chromosomal numbers varying from 16 pairs to 31 pairs. These findings challenge the claim that the accumulation of chromosomal rearrangements drive complete reproductive isolation, and promote equids as a fundamental model for understanding the interplay between chromosomal structure, gene flow, and, ultimately, speciation.

Vultures are scavengers that fill a key ecosystem niche, in which they have evolved a remarkable tolerance to bacterial toxins in decaying meat. Here we report the first deep metagenomic analysis of the vulture microbiome. Through face and gut comparisons of 50 vultures representing two species, we demonstrate a remarkably conserved low diversity of gut microbial flora. The gut samples contained an average of 76 operational taxonomic units (OTUs) per specimen, compared with 528 OTUs on the facial skin. Clostridia and Fusobacteria, widely pathogenic to other vertebrates, dominate the vulture's gut microbiota. We reveal a likely faecal-oral-gut route for their origin. DNA of prey species detectable on facial swabs was completely degraded in the gut samples from most vultures, suggesting that the gastrointestinal tracts of vultures are extremely selective. Our findings show a strong adaption of vultures and their bacteria to their food source, exemplifying a specialized host-microbial alliance.

Recent studies have shown that wild ruminants are sources of previously undescribed microorganisms, knowledge of which can improve our understanding of the complex microbial interactions in the foregut. Here, we investigated the microbial community of seven wild-caught giraffes (Giraffa camelopardalis), three of which were fed natural browse and four were fed Boskos pellets, leafy alfalfa hay, and cut savanna browse, by characterizing the 16S rRNA gene diversity using 454 FLX high-throughput sequencing. The microbial community composition varied according to diet, but differed little between the ruminal fluid and solid fraction. The giraffe rumen contained large levels of the phyla of Firmicutes and Bacteroidetes independent of diet, while Prevotella, Succinclasticium, and Methanobrevibacter accounted for the largest abundant taxonomic assigned genera. However, up to 21% of the generated sequences could not been assigned to any known bacterial phyla, and c. 70% not to genus, revealing that the giraffe rumen hosts a variety of previously undescribed bacteria., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: Establishment of haematological and biochemical reference intervals is important to assess health of animals on individual and population level. Reference intervals for 13 haematological and 34 biochemical variables were established based on 88 apparently healthy free-ranging brown bears (39 males and 49 females) in Sweden. The animals were chemically immobilised by darting from a helicopter with a combination of medetomidine, tiletamine and zolazepam in April and May 2006-2012 in the county of Dalarna, Sweden. Venous blood samples were collected during anaesthesia for radio collaring and marking for ecological studies. For each of the variables, the reference interval was described based on the 95% confidence interval, and differences due to host characteristics sex and age were included if detected. To our knowledge, this is the first report of reference intervals for free-ranging brown bears in Sweden., Results: The following variables were not affected by host characteristics: red blood cell, white blood cell, monocyte and platelet count, alanine transaminase, amylase, bilirubin, free fatty acids, glucose, calcium, chloride, potassium, and cortisol. Age differences were seen for the majority of the haematological variables, whereas sex influenced only mean corpuscular haemoglobin concentration, aspartate aminotransferase, lipase, lactate dehydrogenase, β-globulin, bile acids, triglycerides and sodium., Conclusions: The biochemical and haematological reference intervals provided and the differences due to host factors age and gender can be useful for evaluation of health status in free-ranging European brown bears.