Usefulness and limitations of pollen characters in environmental studies based on Viola L. species (sect. Melanium Ging.)

The aim of these studies was to determine the effect of environmental pollution on pollen development. Pollen heteromorphism (the presence of pollen morphs differing in aperture number in one flower of a plant), pollen viability (stainability) and pollen grain size in European metallophytes from sect. Melanium Ging. (Viola L., Violaceae) were analyzed by SEM and histochemical staining.Plants’ tolerance to heavy metals is positively correlated with their pollen viability, which should be termed stainability as it depends on the staining method applied and is not correlated with pollen germination. Abortive pollen can be produced as an effect of heavy metals but also may result from hybridization, a very common phenomenon in pansies. Pollen stainability in hybrids can be high (even exceeding 70%) or low (barely above 20%), and stainable pollen grains can differ greatly in size (from very small to giant), indicating a cytological imbalance resulting from disturbed meiosis. The number of pollen apertures is an adaptive character in facultative metallophytes. Plants from a metallicolous population produced a wider range of aperture number (3, 4, 5) than plants from a non-metallicolous population, which developed only 4- and 5-aperturate pollen. Three-aperturate longer-lived pollen are favored in the harsh conditions of a metal-polluted environment

Potential of herbariomics for studying repetitive DNA in angiosperms

Repetitive DNA has an important role in angiosperm genomes and is relevant to our understanding of genome size variation, polyploidisation and genome dynamics more broadly. Much recent work has harnessed the power of high-throughput sequencing (HTS) technologies to advance the study of repetitive DNA in flowering plants. Herbarium collections provide a useful historical perspective on genome diversity through time, but their value for the study of repetitive DNA has not yet been explored. We propose that herbarium DNA may prove as useful for studies of repetitive DNA content as it has for reconstructed organellar genomes and low-copy nuclear sequence data. Here we present a case study in the tobacco genus (Nicotiana; Solanaceae), showing that herbarium specimens can provide accurate estimates of the repetitive content of angiosperm genomes by direct comparison with recently-collected material. We show a strong correlation between the abundance of repeat clusters, e.g., different types of transposable elements and satellite DNA, in herbarium collections versus recent material for four sets of Nicotiana taxa. These results suggest that herbarium specimen genome sequencing (herbariomics) holds promise for both repeat discovery and analyses that aim to investigate the role of repetitive DNAs in genomic evolution, particularly genome size evolution and/or contributions of repeats to the regulation of gene space

Genome-wide repeat dynamics reflect phylogenetic distance in closely related allotetraploid Nicotiana (Solanaceae)

Nicotiana sect. Repandae is a group of four allotetraploid species originating from a single allopolyploidisation event approximately 5 million years ago. Previous phylogenetic analyses support the hypothesis of N. nudicaulis as sister to the other three species. This is concordant with changes in genome size, separating those with genome downsizing (N. nudicaulis) from those with genome upsizing (N. repanda, N. nesophila, N. stocktonii). However, a recent analysis reflecting genome dynamics of different transposable element families reconstructed greater similarity between N. nudicaulis and the Revillagigedo Island taxa (N. nesophila and N. stocktonii), thereby placing N. repanda as sister to the rest of the group. This could reflect a different phylogenetic hypothesis or the unique evolutionary history of these particular elements. Here we re-examine relationships in this group and investigate genome-wide patterns in repetitive DNA, utilising high-throughput sequencing and a genome skimming approach. Repetitive DNA clusters provide support for N. nudicaulis as sister to the rest of the section, with N. repanda sister to the two Revillagigedo Island species. Clade-specific patterns in the occurrence and abundance of particular repeats confirm the original (N. nudicaulis (N. repanda (N. nesophila ? N. stocktonii))) hypothesis. Furthermore, overall repeat dynamics in the island species N. nesophila and N. stocktonii confirm their similarity to N. repanda and the distinctive patterns between these three species and N. nudicaulis. Together these results suggest that broad-scale repeat dynamics do in fact reflect evolutionary history and could be predicted based on phylogenetic distance

Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study

Bat diversity in the lowland forests of the Heart of Borneo

Borneo’s rainforests are renowned for their high levels of biodiversity, yet information on the distribution and structure of this diversity is lacking, particularly for less charismatic taxonomic groups. We quantified bat diversity across ten sites within a contiguous tract of largely undisturbed rainforest in the Heart of Borneo (HoB) transboundary conservation area. Using comparative analyses of 1,362 bat captures from six sites in Brunei Darussalam, together with data from four additional sites in neighbouring territories, we show that the main differences in bat assemblage composition between sites were driven by the abundances of a few cave-roosting species. Beta diversity (distance decay) was notably low and non-significant. Bat assemblage structure in these undisturbed palaeotropical forests is therefore relatively homogenous in the absence of environmental gradients. By adding 15 bat species to the Brunei national inventory, we confirm the area of north Borneo to be species-diverse and therefore a priority for conservation efforts. However, we also highlight that coastal forest to be included in a recent extension to the HoB hosts bat assemblages with the fewest species and lowest densities. We maintain that extending the HoB in Brunei to include a more diverse portfolio of habitat types is still warranted on the grounds of maximising botanical diversity and habitat area, as long as it does not detract attention from interior forests that support higher vertebrate diversity

Extensive plastid-nuclear discordance in a recent radiation of Nicotiana section Suaveolentes (Solanaceae)

Nicotiana section Suaveolentes is the largest section of Nicotiana and is a monophyletic group of allotetraploid species. Most of the species are endemic to Australia, but three species occur on islands in the South Pacific as far east as French Polynesia and one species is native to Namibia. Here, we present phylogenetic results based on genome skimming, with near-complete taxon sampling and multiple accessions sampled for several species. These represent the first phylogenetic results for the section that include most recognized taxa, using wild-sourced material wherever possible. Despite known chromosome number and genome size changes in the section, there is little divergence in the ribosomal DNA operon (26S, 18.S and 5.8S plus associated spacers) and plastid genomes, with little to no taxonomic signal in plastome phylogenetic results and clear plastid-nuclear discordance. These results contrast with strong morphological differentiation (both floral and vegetative) between most of the core Australian taxa and obvious differences in ecological preferences. Together, these initial results portray Nicotiana section Suaveolentes as experiencing recent and ongoing radiation in the arid zone of Australia

Conservation importance of limestone karst outcrops for Palaeotropical bats in a fragmented landscape

Limestone karst landscapes are important for biodiversity yet are increasingly threatened by development activities such as mining. Furthermore, karsts are often scattered and isolated by agriculture, and are rarely considered in landscape planning because of a paucity of biodiversity data. We determined the conservation significance of an isolated limestone karst outcrop for insectivorous bats by quantifying the influence of this roosting resource on local assemblage structure across a fragmented landscape in peninsular Malaysia. Using a combination of rank abundance, gradient and randomisation analyses, we demonstrate that bat assemblages at nine forest sites are structured following a spatial gradient of increasing distance from a karst roosting resource. The assemblage at our karst site was dominated by a superabundance of three cave-roosting species, two of which were also found to dominate assemblages up to 11 km away. Cave-roosting bats exhibited a significant decay in abundance related to the distance from karst, with sites closest to karst also characterised by a rarity of tree cavity/foliage-roosting species that were otherwise common. Gradient analysis revealed that differences in assemblage composition were largely associated with the distance from the karst and, to a lesser extent, forest isolation and area. Our findings suggest that isolated karst outcrops can serve as important population reservoirs for cave-roosting bats, which subsidise diversity levels in forest fragments that might otherwise be expected to decline over time. While conservation efforts need to focus on maintaining large areas of connected forest, landscape management needs to ensure protection of karsts as point resources for cave-roosting bats

Existence of a positive feedback loop to activate the <i>Tbx5</i> gene.

<p>(A) An sgRNA was designed to target the sequence (shown in red) immediately downstream of the first methionine codon (ATG in blue). The underlined TGG sequence corresponds to the protospacer adjacent motif (PAM). (B) Obtained internal deletion alleles in a <i>Tbx5</i> null ES cell line. The first methionine codon (ATG) is shown in bold, and the PAM (TGG) is shown in green. (C) Immunoblot analysis of TBX5 and eYFP in differentiated BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP ES cells that were either wild type or null (k/o) for <i>Tbx5</i>. α-Tubulin was examined as a loading control. The whole blotted membrane is indicated as a whole in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140831#pone.0140831.s009" target="_blank">S8 Fig</a>. (D) BAC <i>Tbx5</i>^CreERT2<i>/ROSA26</i>^eYFP/eYFP ES cells either rendered <i>Tbx5</i> null by the CRISPR/Cas9 or left unmodified (WT) were induced to differentiate into cardiomyocytes in the presence of 4-hydroxytamoxifen. The cells were then subjected to flow cytometric analysis of TNNT2 and eYFP expression. ES cells without the BAC transgene were used as control. (E) Representative flow cytometric plots as well as mean ± SEM values from three independent experiments are shown. *<i>P</i> < 0.05; NS, not significant (Student's <i>t</i> test). (F) Schematic representation of the early CPs. CMs; cardiomyocytes, CSCs; electric conduction system cells.</p

Dynamic Changes in the Expression Profiles of CPs from the EB to EHF Stages.

<p>(A) Proportion of single-cell cDNA preparations positive for <i>Mesp1</i>, <i>Myl2</i>, <i>Isl1</i>, and <i>Myl7</i> expression at the indicated embryonic stages as determined by PCR analysis. (B) WISH analysis of <i>Mesp1</i>, <i>Myl2</i>, <i>Isl1</i>, and <i>Myl7</i> expression in the mouse embryo at the EB, LB, and EHF stages. Embryos are shown in the left lateral view. Expression of <i>Mesp1</i> was detected at a low level in the anterior mesoderm at the EB stage. <i>Myl2</i> was not detected as expected by PCR analysis on single cell cDNA preparations in (A). A; anterior, P; posterior.</p

Single-cell expression profiling of the earliest CPs.

<p>(A) WISH analysis of <i>Nkx2-5</i> and <i>Tbx5</i> expression in mouse embryos at EB, late bud (LB), and EHF stages. Embryos are shown in left lateral view. A; anterior, P; posterior, Red arrows; the most anterior part of the embryo. (B) Strategy for generation of single-cell cDNA preparations. (C) Classification of single-cell cDNA preparations of CPs from EB, LB, and EHF stages by PCR analysis of marker genes. The number of preparations is shown in parentheses. (D) Subpopulation shift between EB and Somite stages. (E) Taqman assay for <i>Nkx2-5</i> and <i>Tbx5</i> on constructed single-cell cDNA preparations. (F) Distribution of <i>Nkx2-5</i>-expressing CPs and <i>Tbx5</i>-expressing CPs in EHF stage embryo. The pictures of the embryos in the upper panel indicate whole mount <i>in situ</i> hybridization for <i>Nkx2-5</i> and <i>Tbx5</i> in EHF stage embryos in the frontal view. Note the area of <i>Nkx2-5</i> is wider than that of <i>Tbx5</i>. Fluorescence images indicate the immunostained EHF stage mouse embryo for NKX2-5 and TBX5. The illustration at the upper right panel shows section plane of fluorescence image. D; distal, EN; endoderm, NE; neural ectoderm. Blue; 4ʹ,6-diamidino-2-phenylindole (DAPI), Green; TBX5, Red; NKX2-5, L; left, P; proximal, R; right. Scale bar; 100 μm.</p