Substrate specialisation drives an unexpectedly diverse radiation in barking geckos (Ptenopus: Gekkonidae).

Barking geckos (genus Ptenopus) are terrestrial, burrowing lizards endemic to southern Africa, currently with three recognised species. Two species are range-restricted (P. kochi and P. carpi) and display clear differences in substrate preference (soft sand vs. hard gravel). The third and most widespread species, P. garrulus, occurs on a variety of substrates of differing hardness, across potential geographic barriers, and over a steep climatic gradient. Variations in morphology and advertisement calls indicates that P. garrulus may be a species complex. Two subspecies of P. garrulus are currently recognised: P. g. maculatus and P. g. garrulus. To investigate species boundaries, we produced the first comprehensive phylogeny for the genus. We used a novel application of multiple regression on matrices models to assess multiple environmental drivers of diversification, as contrasted to isolation by distance. We show that P. kochi, P. carpi, and P. g. garrulus are valid species, but that P. g. maculatus is a paraphyletic complex of five previously unrecognised taxa. Specialisation onto different substrates was likely the main driver of divergence, with parapatric occurrence of two to four clades occurring at each of the three substrate transition zones identified a priori. The region encompasses diverse bioclimatic regions and potential geographic barriers, and these likely played a role in some divergence events.

The bii4africa dataset of faunal and floral population intactness estimates across Africa's major land uses.

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species' population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate 'intactness scores': the remaining proportion of an 'intact' reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region's major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems.

De Novo Whole Genome Assemblies for Two Southern African Dwarf Chameleons (Bradypodion, Chamaeleonidae).

A complete and high-quality reference genome has become a fundamental tool for the study of functional, comparative, and evolutionary genomics. However, efforts to produce high-quality genomes for African taxa are lagging given the limited access to sufficient resources and technologies. The southern African dwarf chameleons (Bradypodion) are a relatively young lineage, with a large body of evidence demonstrating the highly adaptive capacity of these lizards. Bradypodion are known for their habitat specialization, with evidence of convergent phenotypes across the phylogeny. However, the underlying genetic architecture of these phenotypes remains unknown for Bradypodion, and without adequate genomic resources, many evolutionary questions cannot be answered. We present de novo assembled whole genomes for Bradypodion pumilum and Bradypodion ventrale, using Pacific Biosciences long-read sequencing data. BUSCO analysis revealed that 96.36% of single copy orthologs were present in the B. pumilum genome and 94% in B. ventrale. Moreover, these genomes boast scaffold N50 of 389.6 and 374.9 Mb, respectively. Based on a whole genome alignment of both Bradypodion genomes, B. pumilum is highly syntenic with B. ventrale. Furthermore, Bradypodion is also syntenic with Anolis lizards, despite the divergence between these lineages estimated to be nearly 170 Ma. Coalescent analysis of the genomic data also suggests that historical changes in effective population size for these species correspond to notable shifts in the southern African environment. These high-quality Bradypodion genome assemblies will support future research on the evolutionary history, diversification, and genetic underpinnings of adaptation in Bradypodion.

When to shed? Patterns and drivers of time to first ecdysis in snakes.

Time from birth or hatching to the first shed (postnatal ecdysis) in snakes ranges from about an hour to several weeks depending upon the species. We assessed patterns in time to postnatal ecdysis in 102 snake species for which we could source appropriate information, covering 2.6% of all extant snake species, and related measures to various biological traits. Reconstruction revealed ancestral time to postnatal ecdysis to be 11 days. Since time to postnatal ecdysis can be shorter or longer than the ancestral state, we argue that there are several competing drivers for time to postnatal ecdysis. A reduced time to postnatal ecdysis has evolved in several lineages, commonly in ambush-foraging, viviparous vipers, while extended time to postnatal ecdysis is associated with oviparous species with maternal care. Of central importance is the impact of postnatal ecdysis on the scent levels of neonates, resulting in a reduction of time to postnatal ecdysis in chemically cryptic species, while the pivotal role of scent in mother-neonate recognition has resulted in the retention or extension of time to postnatal ecdysis. We showed that postnatal ecdysis improves chemical crypsis. The patterns revealed in this study suggest that measures of time to postnatal ecdysis can provide insights into the biology of snakes and be used as an indicator of certain life history traits.

A global reptile assessment highlights shared conservation needs of tetrapods.

Comprehensive assessments of species' extinction risks have documented the extinction crisis1 and underpinned strategies for reducing those risks2. Global assessments reveal that, among tetrapods, 40.7% of amphibians, 25.4% of mammals and 13.6% of birds are threatened with extinction3. Because global assessments have been lacking, reptiles have been omitted from conservation-prioritization analyses that encompass other tetrapods4-7. Reptiles are unusually diverse in arid regions, suggesting that they may have different conservation needs6. Here we provide a comprehensive extinction-risk assessment of reptiles and show that at least 1,829 out of 10,196 species (21.1%) are threatened-confirming a previous extrapolation8 and representing 15.6 billion years of phylogenetic diversity. Reptiles are threatened by the same major factors that threaten other tetrapods-agriculture, logging, urban development and invasive species-although the threat posed by climate change remains uncertain. Reptiles inhabiting forests, where these threats are strongest, are more threatened than those in arid habitats, contrary to our prediction. Birds, mammals and amphibians are unexpectedly good surrogates for the conservation of reptiles, although threatened reptiles with the smallest ranges tend to be isolated from other threatened tetrapods. Although some reptiles-including most species of crocodiles and turtles-require urgent, targeted action to prevent extinctions, efforts to protect other tetrapods, such as habitat preservation and control of trade and invasive species, will probably also benefit many reptiles.

In vivo assessment of glutamine anaplerosis into the TCA cycle in human pre-malignant and malignant clonal plasma cells.

BACKGROUND: Overexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown. METHODS: Human volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labeled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma. RESULTS: Despite notable heterogeneity, stable isotope-resolved metabolomics (SIRM) revealed that the mean 13C-labeled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients. CONCLUSION: Measurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM. TRIAL REGISTRATION: NCT03384108 and NCT03119883.

Correction to: Male energy reserves, mate-searching activities, and reproductive success: alternative resource use strategies in a presumed capital breeder.

The authors would like to correct the error in table 4 which was incorrectly published in original version. Correct version of Table 4 is updated here.

Male energy reserves, mate-searching activities, and reproductive success: alternative resource use strategies in a presumed capital breeder.

Sexual selection studies often focus on morphological traits that are important only in the later stages of mate acquisition. Comparatively little is known about traits that lead to mate acquisition, such as mate-searching activities. We experimentally manipulated body condition (i.e., the energy reserves) in male puff adders (Bitis arietans) prior to the mating season using supplemental feeding in the field, and used radio-telemetry and DNA paternity analyses to characterize the relationships between male energy reserves, mating activities, and reproductive success. We found that male mobility is a strongly sexually selected trait because males that travelled further in search of females have higher female encounter rates and reproductive success. However, supplemental feeding did not have a significant effect on mating activities or reproductive success, because control snakes compensated by foraging more often during the mating season. The time invested in foraging by control snakes did not come at the costs of decreased mating activities or opportunity compared to fed snakes, because the latter spent the spare time resting. Our experimental field research directly demonstrates the link between male mobility and reproductive success, identifying the ultimate mechanism leading to the evolution of prolonged male mate-searching activities in snakes, and indicates that male puff adders, presumed capital breeders, adjust their breeding tactics according to resource availability.

Mortality in Patients With Atrial Fibrillation Receiving Nonrecommended Doses of Direct Oral Anticoagulants.

BACKGROUND: The recommended doses for direct oral anticoagulants (DOACs) to prevent stroke and systemic embolism (SE) in patients with atrial fibrillation (AF) are described in specific regulatory authority approvals. OBJECTIVES: The impact of DOAC dosing, according to the recommended guidance on all-cause mortality, stroke/SE, and major bleeding, was assessed at 2-year follow-up in patients with newly diagnosed AF. METHODS: Of a total of 34,926 patients enrolled (2013 to 2016) in the prospective GARFIELD-AF (Global Anticoagulant Registry in the FIELD-AF), 10,426 patients received a DOAC. RESULTS: The majority of patients (72.9%) received recommended dosing, 23.2% were underdosed, and 3.8% were overdosed. Nonrecommended dosing (underdosage and overdosage combined) compared with recommended dosing was associated with a higher risk of all-cause mortality (hazard ratio [HR]: 1.24; 95% confidence interval [CI]: 1.04 to 1.48); HR: 1.25 (95% CI: 1.04 to 1.50) for underdosing, and HR: 1.19 (95% CI: 0.83 to 1.71) for overdosing. The excess deaths were cardiovascular including heart failure and myocardial infarction. The risks of stroke/SE and major bleeding were not significantly different irrespective of the level of dosing, although underdosed patients had a significantly lower risk of bleeding. A nonsignificant trend to higher risks of stroke/SE (HR: 1.51; 95% CI: 0.79 to 2.91) and major bleeding (HR: 1.29; 95% CI: 0.59 to 2.78) was observed in patients with overdosing. CONCLUSIONS: In GARFIELD-AF, most patients received the recommended DOAC doses according to country-specific guidelines. Prescription of nonrecommended doses was associated with an increased risk of death, mostly cardiovascular death, compared with patients on recommended doses, after adjusting for baseline factors. (Global Anticoagulant Registry in the Field-AF [GARFIELD-AF]; NCT01090362).

Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.

Metabolomic and Lipidomic Profiling of Bone Marrow Plasma Differentiates Patients with Monoclonal Gammopathy of Undetermined Significance from Multiple Myeloma.

Oncogenic drivers of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) such as c-MYC have downstream effects on intracellular metabolic pathways of clonal plasma cells (PCs). Thus, extracellular environments such as the bone marrow (BM) plasma likely have unique metabolite profiles that differ from patients with MGUS compared to MM. This study utilized an untargeted metabolite and targeted complex lipid profiling of BM plasma to identify significant differences in the relative metabolite levels between patients with MGUS and MM from an exploratory cohort. This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles such as decreased branch chain amino acids (BCAAs) and increased catabolism of tryptophan to the active kynurenine metabolite 3-hydroxy-kynurenine between patients with MGUS and MM. A decrease in the total levels of complex lipids such as phosphatidylethanolamines (PE), lactosylceramides (LCER) and phosphatidylinositols (PI) were also detected in the BM plasma samples from MM compared to MGUS patients. Thus, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between patients with MGUS and MM. This may provide insight into the possible differences of the intracellular metabolic pathways of their clonal PCs.

A draft genome sequence of the elusive giant squid, Architeuthis dux.

BACKGROUND: The giant squid (Architeuthis dux; Steenstrup, 1857) is an enigmatic giant mollusc with a circumglobal distribution in the deep ocean, except in the high Arctic and Antarctic waters. The elusiveness of the species makes it difficult to study. Thus, having a genome assembled for this deep-sea-dwelling species will allow several pending evolutionary questions to be unlocked. FINDINGS: We present a draft genome assembly that includes 200 Gb of Illumina reads, 4 Gb of Moleculo synthetic long reads, and 108 Gb of Chicago libraries, with a final size matching the estimated genome size of 2.7 Gb, and a scaffold N50 of 4.8 Mb. We also present an alternative assembly including 27 Gb raw reads generated using the Pacific Biosciences platform. In addition, we sequenced the proteome of the same individual and RNA from 3 different tissue types from 3 other species of squid (Onychoteuthis banksii, Dosidicus gigas, and Sthenoteuthis oualaniensis) to assist genome annotation. We annotated 33,406 protein-coding genes supported by evidence, and the genome completeness estimated by BUSCO reached 92%. Repetitive regions cover 49.17% of the genome. CONCLUSIONS: This annotated draft genome of A. dux provides a critical resource to investigate the unique traits of this species, including its gigantism and key adaptations to deep-sea environments.

Horizon scanning for South African biodiversity: A need for social engagement as well as science.

A horizon scan was conducted to identify emerging and intensifying issues for biodiversity conservation in South Africa over the next 5-10 years. South African biodiversity experts submitted 63 issues of which ten were identified as priorities using the Delphi method. These priority issues were then plotted along axes of social agreement and scientific certainty, to ascertain whether issues might be "simple" (amenable to solutions from science alone), "complicated" (socially agreed upon but technically complicated), "complex" (scientifically challenging and significant levels of social disagreement) or "chaotic" (high social disagreement and highly scientifically challenging). Only three of the issues were likely to be resolved by improved science alone, while the remainder require engagement with social, economic and political factors. Fortunately, none of the issues were considered chaotic. Nevertheless, strategic communication, education and engagement with the populace and policy makers were considered vital for addressing emerging issues.

Modeling open surgery in mice to explore peritoneal damage, carbon dioxide humidification and desmoidogenesis.

BACKGROUND: The exposure of the peritoneum to desiccation during surgery generates lasting damage to the mesothelial lining which impacts inflammation and tissue repair. We have previously explored open abdominal surgery in mice subjected to passive airflow however, operating theatres employ active airflow. Therefore, we sought an engineering solution to recapitulate the active airflow in mice. Similarly, to the passive airflow studies we investigated the influence of humidified-warm carbon dioxide (CO2) on this damage in the context of active airflow. Additionally, we addressed the controversial role of surgery in exacerbating desmoidogenesis in a mouse model of familial adenomatous polyposis. METHODS: An active airflow mouse-operating module manufactured to produce the equivalent downdraft airflow to that of a modern operating theatre was employed. We quantified mesothelial cell integrity by scanning electron microscopy (SEM) sampled from the peritoneal wall that was subjected to mechanical damage or not, with and without the delivery of humidified-warm CO2. To explore the role of open and laparoscopic surgery in the process of desmoidogenesis we crossed Apcmin/ + C57Bl/6 mice with p53 +/- mice to generate animals that developed desmoid tumors with 100% penetrance. RESULTS: One hour of active airflow generates substantial damage to peritoneal mesothelial cells and their microvilli as measured at 24 h post intervention, which is significantly greater than that generated by passive airflow. Use of humidified-warm CO2 mostly protects the mesothelium that had not experienced additional mechanical (surgical) damage at 24 h. Maximal damage was evident in all treatment groups regardless of flow or use of gas. At day 10 mechanically-damaged peritoneum remains in mice but is essentially repaired in the gas-treated groups. Regarding desmoidogenesis, operating procedures did not increase the frequency of desmoid tumors but their frequency correlated with time following surgery but not age of mice. CONCLUSIONS: Active airflow generates more peritoneal damage than passive airflow and is reduced significantly by the use of humidified-warm CO2. Introduced peritoneal damage is largely repaired in mice by day 10 with gas. Desmoid tumor incidence is not increased substantially by surgery itself but rises over time following surgery compared to non-surgery mice.

The cutting-edge morphology of the mole snake's dental apparatus.

The mole snake (Pseudaspis cana) is capable of inflicting unusual bites in defence and during male combat that present as two parallel lacerations. We investigated the dental morphology of the mole snake by making SEM images, and by CT-scanning and digitally reconstructing the skulls of 14 specimens comprising both sexes. The lengths, volumes, shapes and positions of maxillary and dentary teeth were compared within individuals, between individuals, and between sexes. CT reconstructions show the occurrence of large, flat triangular teeth at the posterior end of the maxilla that are angled to point towards the posterior of the skull. SEM imagery highlights the presence of sharp ridges (carinae) on the posterior edges of the posterior dentary and maxillary teeth. Males have greater dental specialization, maxillary tooth variation, enlargement of the posterior-most maxillary teeth, and dentary teeth with posterior carinae. We hypothesize that mole snake dental specializations are adaptations for their particular form of male combat and possibly for subduing prey in the confines of underground burrows. Our findings reveal a complex dental morphology in mole snakes and provide impetus for further studies on the functional morphology of snake teeth.

Foraging mode, relative prey size and diet breadth: A phylogenetically explicit analysis of snake feeding ecology.

Foraging modes (ambush vs. active foraging) are often correlated with a suite of morphological, physiological, behavioural and ecological traits known as the "adaptive syndrome" or "syndrome hypothesis." In snakes, an ecological correlate often reported in the literature is that ambush-hunting snakes have a higher relative meal size compared to actively foraging snakes which feed on smaller prey items. This "large meal versus small meal" feeding hypothesis between ambush and active foragers has become a widely accepted paradigm of snake feeding ecology, despite the fact that no rigorous meta-analysis has been conducted to support this generalization. We conducted a phylogenetically explicit meta-analysis, which included ca. 100 species, to test this paradigm of snake feeding ecology. We gathered data on prey size by inducing regurgitation by palpation in free-ranging snakes and by examining the stomach contents of preserved museum specimens. When we found prey, we recorded both snake and prey mass to estimate relative prey mass (prey mass/snake mass). We also reviewed published studies of snake feeding ecology to gather similar information for other species. Ambush and active foragers did not differ in minimum or average meal size but the maximum meal sizes consumed by ambush-foraging snakes were larger than the maximum meal sizes eaten by active foragers. This results in ambush-foraging snakes consuming a significantly wider range of meal sizes, rather than being large meal specialists compared to active foragers. We argue that ambush foragers evolved to be more opportunistic predators because they encounter prey less frequently compared to active foragers. This hypothesis is further supported by the fact that ambush foragers also exhibited marginally wider diet breadths, consuming a broader range of prey types in comparison with active foragers. Our study challenges aspects of the foraging syndrome as it is currently conceived, and our results have important implications for our understanding of how foraging mode has shaped the behaviour and physiology of ambush-foraging snakes.

Diversifying into the branches: Species boundaries in African green and bush snakes, Philothamnus (Serpentes: Colubridae).

The African green and bush snakes of the genus Philothamnus currently comprises 21 species and three subspecies and occurs throughout sub-Saharan Africa. The genus has been the subject of previous taxonomic revisions based on traditional morphological characters and limited genetic assessment, and may not reflect their evolutionary history. Indeed, previous findings based on phylogenetics show discordant results of interspecific relationships and question the monophyly of the genus, although taxon sampling has been limited to date. We investigated phylogenetic affinities within Philothamnus with more inclusive genetic and geographical sampling, with the aim of better understanding their evolutionary history, so that future taxonomic revision of Philothamnus can be better informed. Species relationships were examined within a phylogenetic context and sampling included 133 ingroup samples from 16 taxa. Phylogenies were constructed in Bayesian and likelihood frameworks using three mitochondrial (16S, cyt b and ND4) and two nuclear (c-mos and RAG1) markers. Competing hypotheses relating to the monophyly of the genus were tested with a Shimodaira-Hasegawa test. To examine species boundaries, Bayesian General Mixed Yule-Coalescent Model and multi-rate Poisson Tree Processes analyses were conducted. In addition, a barcoding approach was used to further clarify species-level relationships by comparing frequency distributions between intra- and interspecific sequence divergence. The genus was recovered as monophyletic; however, species-delimitation results suggest that the current taxonomy does not reflect the evolutionary history of this group. For example, Philothamnus s. semivariegatus is paraphyletic, with at least four distinct clades. Philothamnus carinatus consists of two cryptic (sister) lineages from Central and West Africa that are deeply divergent, suggesting a long history of isolation between those regions. Furthermore, the subspecies P. n. natalensis and P. n. occidentalis show strong support for species-level divergence, which reflects their morphological and ecological differences. Accordingly, we elevate P. occidentalisnov. comb. to a full species. A fully informed taxonomic revision of these taxa will require additional morphological and ecological data for corroboration, but it seems that the morphological characters (e.g. scalation, dentition) used to describe these species to date are labile within and between species. This most likely has clouded our understanding of the species boundaries within the genus. Our phylogeny and species-delimitation analyses should provide a sounder framework for taxonomy, but may also prove useful toward understanding the morphological adaptations of these species to their respective habitats.

The impact of posture and basking orientation on thermoregulation in the Sungazer (Smaug giganteus).

Body temperature (Tb) is one of the most influential factors affecting physiological processes in ectothermic animals. Reptiles use behaviours such as shuttling, and postural and orientation adjustments to ensure that Tb remains close to a target. It is thought that target body temperature (Ttarget) is aligned to the physiological and behavioural thermal optima of the animal. The Sungazer (Smaug giganteus), a Threatened lizard species, is unique amongst the Cordylidae in that lizards inhabit self-excavated burrows in open grasslands, a habitat in which there are few exposed rock basking sites. In this situation, Tbs are likely to be greatly influenced by postural and orientation adjustments during basking. We measured Tbs using modified iButtons, and calculated Ttarget of free-ranging Sungazers in order to assess the impact of body posture and orientation in the thermoregulation of the species. We used camera traps to record lizard behaviour at burrow entrances and aligned these measures to measures of Tb. Ttarget was 30 ±â€¯1.4 °C, and Tbs remained within this range for 371 ±â€¯162 min per day. The anterior body-up postures resulted in faster heating and higher Tbs. Lizards heated faster when facing away from the sun and spent proportionally more time in this orientation in the morning when Tbs were lower than Ttarget. Our findings suggest that their wide behavioural repertoire allows Sungazers to thermoregulate effectively over a broad range of environmental conditions and may thus be able to effectively respond to changing climatic conditions.