Population structure in Mixornis tit-babblers across Sunda Shelf matches interfluvia of paleo-rivers.

Rivers constitute an important biogeographic divide in vast areas of tropical rainforest, such as the Amazon and Congo Basins. Southeast Asia's rainforests are currently fragmented across islands divided by sea, which has long obscured their extensive history of terrestrial connectivity as part of a vast (but now submerged) subcontinent - Sundaland - during most of the Quaternary. The role of paleo-rivers in determining population structure in Sundaic rainforests at a time when these forests were connected remains little understood. We examined the coloration of museum skins and used the genomic DNA of museum samples and freshly-collected blood tissue of a pair of Sundaic songbird species, the pin-striped and bold-striped tit-babblers (Mixornis gularis and M. bornensis, respectively), to assess the genetic affinity of populations on small Sundaic islands that have largely been ignored by modern research. Our genomic and morphological results place the populations from the Anambas and Natuna Islands firmly within M. gularis from the Malay Peninsula in western Sundaland, even though some of these islands are geographically much closer to Borneo, where M. bornensis resides. Our results reveal genetic structure consistent with the course of Sundaic paleo-rivers and the location of the interfluvia they formed, and add to a small but growing body of evidence that rivers would have been of equal biogeographic importance in Sundaland's former connected forest landscape as they are in Amazonia and the Congo Basin today.

Historic samples reveal loss of wild genotype through domestic chicken introgression during the Anthropocene.

Human activities have precipitated a rise in the levels of introgressive gene flow among animals. The investigation of conspecific populations at different time points may shed light on the magnitude of human-mediated introgression. We used the red junglefowl Gallus gallus, the wild ancestral form of the chicken, as our study system. As wild junglefowl and domestic chickens readily admix, conservationists fear that domestic introgression into junglefowl may compromise their wild genotype. By contrasting the whole genomes of 51 chickens with 63 junglefowl from across their natural range, we found evidence of a loss of the wild genotype across the Anthropocene. When comparing against the genomes of junglefowl from approximately a century ago using rigorous ancient-DNA protocols, we discovered that levels of domestic introgression are not equal among and within modern wild populations, with the percentage of domestic ancestry around 20-50%. We identified a number of domestication markers in which chickens are deeply differentiated from historic junglefowl regardless of breed and/or geographic provenance, with eight genes under selection. The latter are involved in pathways dealing with development, reproduction and vision. The wild genotype is an allelic reservoir that holds most of the genetic diversity of G. gallus, a species which is immensely important to human society. Our study provides fundamental genomic infrastructure to assist in efforts to prevent a further loss of the wild genotype through introgression of domestic alleles.

Genomes From Historic DNA Unveil Massive Hidden Extinction and Terminal Endangerment in a Tropical Asian Songbird Radiation.

Quantifying the magnitude of the global extinction crisis is important but remains challenging, as many extinction events pass unnoticed owing to our limited taxonomic knowledge of the world's organisms. The increasing rarity of many taxa renders comprehensive sampling difficult, further compounding the problem. Vertebrate lineages such as birds, which are thought to be taxonomically well understood, are therefore used as indicator groups for mapping and quantifying global extinction. To test whether extinction patterns are adequately gauged in well-studied groups, we implemented ancient-DNA protocols and retrieved whole genomes from the historic DNA of museum specimens in a widely known songbird radiation of shamas (genus Copsychus) that is assumed to be of least conservation concern. We uncovered cryptic diversity and an unexpected degree of hidden extinction and terminal endangerment. Our analyses reveal that >40% of the phylogenetic diversity of this radiation is already either extinct in the wild or nearly so, including the two genomically most distinct members of this group (omissus and nigricauda), which have so far flown under the conservation radar as they have previously been considered subspecies. Comparing the genomes of modern samples with those from roughly a century ago, we also found a significant decrease in genetic diversity and a concomitant increase in homozygosity affecting various taxa, including small-island endemics that are extinct in the wild as well as subspecies that remain widespread across the continental scale. Our application of modern genomic approaches demonstrates elevated levels of allelic and taxonomic diversity loss in a songbird clade that has not been listed as globally threatened, highlighting the importance of ongoing reassessments of extinction incidence even across well-studied animal groups. Key words: extinction, introgression, white-rumped shama, conservation.

Integrating voice and phenotype in a revision of the brush cuckoo Cacomantis variolosus (Aves: Cuculidae) complex.

Brush cuckoos of the Cacomantis variolosus complex, which range from Southeast Asia to Australia and the Solomon Islands, have undergone much taxonomic upheaval. Here we examine 389 vocal recordings, 832 skins, and records of brood parasitism and habitat partitioning to shed light on their species and subspecies taxonomy. Bioacoustic analysis revealed seven distinct vocal groups. Among morphological markers, shape and proportions of the tail were found to be supporting indicators, in addition to plumage tone and pattern. Integration of the resulting data set distinguished six species-level taxa within the complex: sepulcralis in the Philippines and Sundaland east to central Nusa Tenggara, virescens in Sulawesi and the Sula Archipelago, aeruginosus in the Moluccas, variolosus in east Nusa Tenggara, the Moluccas, north and east Australia, New Guinea and Bismarck Archipelago, blandus in the Admiralty Islands, and addendus in the Solomon Islands. Our review of infra-specific differentiation among species leads us to distinguish 13 subspecies. All taxa identified are listed in a summary classification of the complex. Taxon diversity is greatest in the Moluccas, where two habitat-partitioned species and five allopatric subspecies occur, of which one species and three subspecies are endemic to this region.

In vivo identification and validation of novel potential predictors for human cardiovascular diseases.

Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such causative candidate genes, often preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of human heart phenotype-associated GWAS candidates with little or no apparent connection to cardiac function. Building on the conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients impacting on the heart rate in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.

Historic and modern genomes unveil a domestic introgression gradient in a wild red junglefowl population.

The red junglefowl Gallus gallus is the ancestor of the domestic chicken and arguably the most important bird species on Earth. Continual gene flow between domestic and wild populations has compromised its gene pool, especially since the last century when human encroachment and habitat loss would have led to increased contact opportunities. We present the first combined genomic and morphological admixture assessment of a native population of red junglefowl, sampled from recolonized parts of its former range in Singapore, partly using whole genomes resequenced from dozens of individuals. Crucially, this population was genomically anchored to museum samples from adjacent Peninsular Malaysia collected ~110-150 years ago to infer the magnitude of modern domestic introgression across individuals. We detected a strong feral-wild genomic continuum with varying levels of domestic introgression in different subpopulations across Singapore. Using a trait scoring scheme, we determined morphological thresholds that can be used by conservation managers to successfully identify individuals with low levels of domestic introgression, and selected traits that were particularly useful for predicting domesticity in genomic profiles. Our study underscores the utility of combined genomic and morphological approaches in population management and suggests a way forward to safeguard the allelic integrity of wild red junglefowl in perpetuity.

A lost world in Wallacea: Description of a montane archipelagic avifauna.

Birds are the best-known animal class, with only about five or six new species descriptions per year since 1999. Integrating genomic and phenotypic research with arduous fieldwork in remote regions, we describe five new songbird species and five new subspecies from a small area near Sulawesi, Indonesia, all collected in a single 6-week expedition. Two factors contributed to the description of this large number of species from such a small geographic area: (i) Knowledge of Quaternary Period land connections helped pinpoint isolated islands likely to harbor substantial endemism and (ii) studying accounts of historic collectors such as Alfred Wallace facilitated the identification of undercollected islands. Our findings suggest that humans' understanding of biogeographically complex regions such as Wallacea remains incomplete.

Controllable atomic scale patterning of freestanding monolayer graphene at elevated temperature.

We show that by operating a scanning transmission electron microscope (STEM) with a 0.1 nm 300 kV electron beam, one can sculpt free-standing monolayer graphene with close-to-atomic precision at 600 °C. The same electron beam that is used for destructive sculpting can be used to image the sculpted monolayer graphene nondestructively. For imaging, a scanning dwell time is used that is about 1000 times shorter than for the sculpting. This approach allows for instantaneous switching between sculpting and imaging and thus fine-tuning the shape of the sculpted lattice. Furthermore, the sculpting process can be automated using a script. In this way, free-standing monolayer graphene can be controllably sculpted into patterns that are predefined in position, size, and orientation while maintaining defect-free crystallinity of the adjacent lattice. The sculpting and imaging processes can be fully computer-controlled to fabricate complex assemblies of ribbons or other shapes.

TEM study of locally coated nanopore fabricated by ion-beam-induced deposition in a thin membrane.

We studied the formation of locally coated sub-10-nm nanopores fabricated by ion-beam milling and ion-beam-induced deposition (IBID) in a thin silicon nitride membrane. Two typical precursor gases representing conductive ((CH(3))(3)Pt(CpCH(3)), CPC for short) and insulating (tetra ethyl oxysilane, TEOS for short) material deposition are used. Three-dimensional electron tomography, EDX and EELS analysis are used to measure the changes in chemical composition and shape of the pores after their formation and at various stages of pore shrinkage. The formation and shrinkage are shown to be due to a shifting competition between IBID and material sputtering during ion-beam exposure. The chemical distribution at the rim of the nanopore is dependent on the precursor gases used: CPC forms a thin carbon layer with small embedded Pt particles at the top and inner surfaces of the nanopore, whereas TEOS forms SiO(x)C(y) with Ga particles dispersed at the rim of the nanopore.

Controlling nanopore size, shape and stability.

Solid-state nanopores are considered a promising tool for the study of biological polymers such as DNA and RNA, due largely to their flexibility in size, potential in device integration and robustness. Here, we show that the precise shape of small nanopores (approximately 5 nm diameter in 20 nm SiN membranes) can be controlled by using transmission electron microscope (TEM) beams of different sizes. However, when some of these small nanopores are immersed in an aqueous solution, their resistance is observed to decrease over time. By comparing nanopores of different shapes using (scanning) TEM both before and after immersion in aqueous solution, we demonstrate that the stability of small nanopores is related to their three-dimensional geometry, which depends on the TEM beam size employed during pore fabrication. Optimal stability is obtained using a TEM beam size of approximately the same size as the intended nanopore diameter. In addition, we show that thermal oxidation can serve as a means to independently control nanopore size following TEM fabrication. These observations provide key guidelines for the fabrication of stable solid-state nanopores on the scale of nucleic acids and small proteins.

Control of shape and material composition of solid-state nanopores.

Solid-state nanopores fabricated by a high-intensity electron beam in ceramic membranes can be fine-tuned on three-dimensional geometry and composition by choice of materials and beam sculpting conditions. For similar beam conditions, 8 nm diameter nanopores fabricated in membranes containing SiO(2) show large depletion areas (70 nm in radius) with small sidewall angles (55 degrees ), whereas those made in SiN membranes show small depletion areas (40 nm) with larger sidewall angles (75 degrees ). Three-dimensional electron tomograms of nanopores fabricated in a SiO(2)/SiN/SiO(2) membrane show a biconical shape with symmetric top and bottom and indicate a mixing of SiN and SiO(2) layers up to 30 nm from the edge of nanopore, with Si-rich particles throughout the membrane. Electron-energy-loss spectroscopy (EELS) reveals that the oxygen/nitrogen ratio near the pore depends on the beam sculpting conditions.

Cross-section transmission electron microscopy characterization of the near-surface structure of medical Nitinol superelastic tubing.

The application of Nitinol in a wide variety of medical implants is progressively increasing because of its unique mechanical properties, durability and biocompatibility. However, as Nitinol consists of about 50 at.% of toxic Ni, certain applications are still hindered by the concern of free Ni release in the surrounding tissue. The latter is controlled by the structure of near-surface layers and can be strongly affected by various surface treatments. A proper application of advanced cross-section sample preparation techniques allows us to characterize the Nitinol near-surface structure down to the nanoscale by means of transmission electron microscopy (TEM). Elemental maps of the Ti, O and Ni distribution, concentration profiles, quantification of composition as well as atomic resolution images at the surface of a Nitinol tubing are presented and the results obtained with different sample preparation and analytical characterization techniques are compared. In addition to a strong decrease of Ni towards the surface of the oxide layer and a Ti depleted layer underneath the oxide, also a possible transformation from TiO to TiO(2) is documented.

Experimental observation of nonlinear ionic transport at the nanometer scale.

Nanometer-sized electrodes are used to probe the transport of ions in liquid by monitoring heterogeneous electrochemical reactions. We observe pronounced nonlinearities of ion flux versus concentration when transport is localized within a region smaller than 10 nm. We show that these observations cannot be explained using conventional continuum, mean-field descriptions of ionic transport. The data indicate that these deviations are caused by the high flux of charged species that is achieved at nanometer-sized electrodes.

Salt dependence of ion transport and DNA translocation through solid-state nanopores.

We report experimental measurements of the salt dependence of ion transport and DNA translocation through solid-state nanopores. The ionic conductance shows a three-order-of-magnitude decrease with decreasing salt concentrations from 1 M to 1 muM, strongly deviating from bulk linear behavior. The data are described by a model that accounts for a salt-dependent surface charge of the pore. Subsequently, we measure translocation of 16.5-mum-long dsDNA for 50 mM to 1 M salt concentrations. DNA translocation is shown to result in either a decrease ([KCl] > 0.4 M) or increase of the ionic current ([KCl] < 0.4 M). The data are described by a model where current decreases result from the partial blocking of the pore and current increases are attributed to motion of the counterions that screen the charge of the DNA backbone. We demonstrate that the two competing effects cancel at a KCl concentration of 370 +/- 40 mM.

Fabrication and characterization of nanopore-based electrodes with radii down to 2 nm.

We report on the fabrication and characterization of gold nanoelectrodes with carefully controlled nanometer dimensions in a matrix of insulating silicon nitride. A focused electron beam was employed to drill nanopores in a thin silicon nitride membrane. The size and shape of the nanopores were studied with high-resolution transmission electron microscopy and electron-energy-loss two-dimensional maps. The pores were subsequently filled with gold, yielding conically shaped nanoelectrodes. The nanoelectrodes were examined by atomic and electrostatic force microscopy. Their applicability in electrochemistry was demonstrated by steady-state cyclic voltammetry. Pores with a radii down to 0.4 nm and electrodes with radii down to 2 nm are demonstrated.