Effect of dark-incubation and inhibitor treatments on size and distribution of charasomes stained by fluorescent FM1-43 in branchlet internodal cells of Chara australis. Under standard conditions of 14/10 h light/darkness, extended charasome-rich areas (A) alternate with small, charasome-poor or -free regions ( B, and smaller magnification C). The white arrow in (C) indicates the neutral line, while the arrow head points to an FM-stained epiphyte. Panels (D) and (E) show different magnifications of cells exposed to continuous darkness for 12 days (control for G–J). Charasomes are mostly small and uniformly distributed (D), but “islands” with larger charasomes occasionally remained (F). (G–J) Effect of 1 μM ikarugamycin (IKA; G), 1.5 μM filipin (H), and of a combination of both inhibitors (I, J) on dark-treated cells.

Apr 30, 2014. For example, estimates of the palaeoaltimetry (18O/16O isotope-based ratios) of the Lunpola basin, in the centre of the QTP, indicate that this part of Tibet. The air circulation massively altered by the rise of the Tianshan and the Higher Himalayas, together with a worldwide cooling since the Middle. Apr 24, 2012. Stone martens (Martes foina) are documented as generalist throughout their distributional range whose diet composition is affected by food availability. We tested if this occurs and what feeding strategies it follows in a typical Mediterranean ecosystem in Central Greece by analysing contents from 106.

Note the extended charasome-rich regions (J). Bars are 10 μm (A,B,D,E,G–I) and 50 μm (C,F,J). Coated Vesicles Are Involved in Charasome Degradation These findings prompted us to inspect the fine structure of dark-exposed cells, in order to decipher the mechanism(s) of charasome degradation. Figure shows an electron micrograph of a branchlet internodal cell, exposed to standard light/dark conditions. The charasomes consist of a complex meshwork of anastomosing tubules formed by plasma membrane invaginations.

CPs and CVs with an average inner diameter of 65.7 ± 11.0 nm ( n = 77) were occasionally seen at the smooth plasma membrane between charasomes, but rarely at the cytoplasmic surface of charasomes, indicating that these organelles were no longer growing and can thus be called “mature” (compare;; ). Charasomes of cells exposed for 1 or 2 days to continuous darkness looked similar to control cells.

However, after 3 days the degradation was clearly visible, and we detected up to four CPs per thin section at the inner surface of charasomes (Figures ). These CPs eventually pinched off as CVs with an inner diameter between 51.4 and 110.9 nm (70.9 ± 11.7 nm on average, n = 30), as suggested by their presence in the immediate surroundings of the CPs (Figures, white arrowheads). The morphology of this coat showed striking similarity to the typical clathrin lattice. The average size of clathrin CVs measured in the present study (around 60–70 nm) is within the range commonly reported in the literature: 50–60 nm in Chara (), 84–91 nm in carrot cells (), 80 nm in pea cotyledons (), 60–100 nm in epithelial cells (). However, our values are larger than the size of CVs found by in the apex of Chara rhizoids (30 nm). It is likely that these differences in size are due to different protocols used for the preparation of electron microscopy samples (e.g., chemical fixation vs.

High pressure freezing and cryo-substitution). Abundance of CPs and CVs varied between cells, cell fragments, and even between charasomes within one fragment. This is consistent with results obtained from AM4-65/FM1-43FX- in vivo staining, showing that charasome degradation does not occur uniformly within one cell (Figure ).

Bubble-like charasomes with few or no inner tubules were characteristic for late stages of charasome degradation (Figure ), and perhaps corresponded to the “hollow” charasomes observed with CLSM (Figure ). Fine structure of charasomes in a control cell (A) and in cells exposed to continuous darkness for 3 days (B–F). (A) Longitudinally sectioned small charasomes (thick black arrows) in a cell exposed to standard light/dark conditions. CV close to a nascent CP (thin black arrow) is seen at the plasma membrane (Ch, chloroplast). (B,C) Longitudinal sections of dark-incubated cells: CPs and CVs (white arrow heads) are detectable at charasomes (thick black arrows). The thin black arrow indicates a CP at the smooth plasma membrane.

Note: there are numerous smooth vesicles near the charasome, some of them containing a central core (black arrow head in C) (g, glycosome). (D) Numerous CPs and CVs (white arrow heads) are present at this tangentially sectioned charasome (thick black arrow). The black arrow head points to a charasome tubule containing a central core. (m, mitochondrion) (E,F) Late stages of darkness-induced charasome degradation. CPs (white arrow heads) formed at the surface of a tangentially sectioned charasome (star in E; note absence of inner tubules) and at a longitudinally sectioned charasome (thick black arrow in F). Note the characteristic polygonal lattice of the clathrin coat in (D,E) (upper white arrow heads).

Bars are 500 nm. Electron micrographs of branchlet internodal cells treated with solvent (A) or ikarugamycin (B–E). (A) Smooth plasma membrane in a cell dark-incubated for 6 days in the presence of 0.05% DMSO (control condition). Thick black arrows indicate remnants of charasomes. (Ch, chloroplast; g, glycosome).

(B–D) Cortical cytoplasm in cells dark-incubated for 6 days in the presence of 0.5 μM ikarugamycin (IKA). Large charasomes (thick black arrows) and CPs or CVs (white arrow heads) at the charasomal surface. Typical CPs are present at the smooth plasma membrane (thin black arrow in D) (m, mitochondrion).

(E) No effect of IKA (4 μM for 30 min) on the formation of CVs (arrow heads) from the TGN (MVB, multivesicular body). Bars are 500 nm. We further studied the fine structure of cells treated with the sterol complexing dye filipin. Supplementary Figure shows the effect of filipin on the cortical cytoplasm of Chara internodes. After 30 min incubation in 1.5 μM filipin, followed by conventional fixation of cells for electron microscopy, plasma membrane, charasome tubules, and membranes of other organelles had a fuzzy appearance (Supplementary Figure; compare to ).

Similar images were obtained when cells were not pretreated with filipin but fixed in the presence of filipin (Supplementary Figure ). In both cases, charasomes and plasma membrane appeared to be more affected than the membrane of mitochondria and chloroplasts. This may indicate higher concentrations of sterols in these compartments. Neither CPs nor CVs could be identified on images taken from thin sections of filipin-treated or filipin-fixed material.

The participation of clathrin in charasome degradation was further supported by experiments using immunofluorescence and an antibody against CHCs. Schmoelzer and co-worker identified filipin as a suitable fluorescent marker for charasomes in 2011. Thus, we used it in this study to visualize charasomes after immunolabeling. Under standard light/dark conditions, the distribution of charasomes is independent of the regions recognized by anti-clathrin (Figures ). However, after 3 days of incubation in darkness, colocalizations of filipin-stained charasomes and clathrin-positive patches were frequently observed (Figures ). Scatter plots and correlation coefficients for both treatments are shown in Supplementary Figure.

Identification, Cloning, and Phylogenetic Analysis of Clathrin Proteins in Chara australis The data presented above indicate a central role of clathrin in charasome degradation. This encouraged us to search for clathrin homologs in the transcriptome of Chara. Databases of C. Australis sequences were created using 454 as well as Illumina RNA sequencing. These databases were searched for clathrin-like sequences.

The obtained results revealed homologous proteins of CHC as well as CLC. Two heavy chains were cloned, sequenced, and named CaCHC1 (1700 amino acids, calculated MW of 192.4 kDa; accession number KX555239) and CaCHC2 (1709 amino acids, calculated MW of 193.6 kDa; accession number KX555240). While the two well known CHC proteins of Arabidopsis thaliana (AtCHC1: At3g11130, accession number Q0WNJ6; AtCHC2: At3g08530, accession number Q0WLB5) showed 97% sequence homology when compared to each other, the two CHCs of C.

Australis shared only 75% identity (see Figure ). Furthermore, CaCHC1 showed higher sequence similarities to CHC of all other plant species investigated than CaCHC2.

For example, compared to A. Thaliana, CaCHC1 revealed 79% homology to AtCHC1 as well as AtCHC2, while CaCHC2 showed only 73%. Figure illustrates the arrangement of conserved domains along the amino acid sequences. AtCHC1 protein is shown as example of known plant CHCs.

The arrangement of domains on CaCHC proteins is comparable to AtCHC. However, CaCHC2 showed a specialty at the C-terminus: a weak but measurable similarity with a bindin domain was detected on the last about 100 amino acids. Clathrin heavy chain proteins in Chara australis.

(A) Protein sequence alignment of clathrin heavy chain (CaCHC1 and CaCHC2) proteins from Chara australis performed with ClustalW. Identical residues are highlighted in black. Numbering of amino acid residues begins at the first methionine.

(B) Arrangement of domains on CHC proteins: AtCHC1 ( Arabisopsis thaliana); CaCHC1, CaCHC2 ( C. Conserved domains are displayed in different colors as indicated. Pfam database classifications of annotated domains are given in brackets. Note the unique bindin-like domain on CaCHC2. (C) Western blot of C. Australis protein extract. The prominent CaCHC band (arrow) was visualized using an antibody against plant CHCs.

Marker = molecular mass marker. Moreover, CHC proteins were visualized by western blot analyses in C. Australis protein extracts. The polyclonal CHC 1,2 antibody (Agrisera AS10 690) was used (Figure ) for detection. A prominent band was visible slightly below marker band 250 kDa, as expected from the calculated molecular mass of the proteins of about 193 kDa. In order to classify CaCHC proteins under the family of CHCs, phylogenetic analyses were performed. As shown in Figure, the 63 aligned CHC protein sequences were divided into several major groups: fungi, green algae, Charophyta, ferns and mosses, and higher plants.

Fungal CHC proteins formed a separate clade (clade F), and therefore presented the outgroup in this phylogenetic analysis. The plant CHC sequences were divided into two major groups (red arrow in Figure ), where the green algae CHCs were sister to the group of Charophyta, as well as all other plant sequences.

Clade C (containing CaCHC1 and CaCHC2) was combined with groups including land plant sequences (ferns, mosses, and higher plants), instead of being classified into the group of other green algae (green box in Figure ). Interestingly, the CHC protein sequence of Klebsormidium flaccidum did not cluster within clade C, but showed sister relations to both sequences of C. Australis and other land plants. In addition to CHC proteins we also had a look at CaCLCs. Several CaCLC sequences were detected, cloned, and sequenced. Protein sequence alignments revealed two groups of highly similar proteins: CaCLC1 and CaCLC2.

CaCLC1 comprises three expressed variants: CaCLC1a (273 amino acids, calculated MW of 28.8 kDa; accession number KX951953), CaCLC1b (283 amino acids, calculated MW of 29.9 kDa; accession number KX951954), and CaCLC1c (280 amino acids, calculated MW of 29.7 kDa; accession number KX951955). CaCLC2 comprises two expressed variants: CaCLC2a (272 amino acids, calculated MW of 30.0 kDa; accession number KX951956) and CaCLC2b (218 amino acids, calculated MW of 23.6 kDa; accession number KX951957).

Protein sequence alignments of all CaCLCs are shown in Figure. CLC proteins are variable in their amino acid composition. Thaliana CLCs (AtCLC) revealed only 37–49% protein sequence homology when compared to each other, while CaCLCs shared 26–93% homology. CaCLC1 proteins shared 25–32% similarity with AtCLCs and 24–28% to Selaginella moellendorfii CLCs. The two CaCLC2 proteins possessed 71% of identical amino acids and share 22–26% similarity to A. Autocad 2012 (64 Bits) + Keygen French.

Thaliana CLCs and 21–25% to S. Amino acid sequence identities were calculated with the bioinformatic software CloneManager ().

Protein sequence alignment of clathrin light chain proteins from Chara australis. Protein sequence alignments of CaCLC1 and CaCLC2 variants were performed with ClustalW (for two sequences) and ClustalOmega (for more than two sequences).

Panel (A) shows the alignment of CaCLC1 variants, Panel (B) shows the alignment of CaCLC2 variants, and the alignment of all variants is displayed in (C). Identical residues are highlighted in black and the conserved clathrin light chain domain (pfam01086) is displayed in green. Numbering of amino acid residues begins at the first methionine.

• • Maps of the region of the Qinghai-Tibetan Plateau (QTP). (A) Four hotspots of biodiversity (green areas) surrounding the QTP: (1) mountains of Central Asia; (2) Himalayas; (3) Indo-Burma; (4) Hengduanshan. Sedimentary basins from which data contributed to estimate the palaeoelevation history of the QTP are shown by purple circles: (a) Junggar; (b) Turpan; (c) Tarim; (d) Qaidam; (e) Xining; (f) Nima; (g) Lunpola; (h) Zhada; (i) Thakkhola; (j) Gyirong; (k) Namling. The yellow line with triangles delineates a region receiving relatively low precipitation (600 mm/year or less). (B) Major mountain ranges (red areas), mountain summits exceeding 8000 m above sea level (black triangles) and main rivers in the region of the QTP. The grey scale indicates the average local altitude (m). These maps were created using WORLDCLIM data (Hijmans et al., ).

• • Conceptual schematics of site location, environmental conditions at sampling sites, and phylogenetic predictions associated with different hypotheses on colonisation and ecological diversification. The upper diagrams show how selected environmental conditions may change with altitude along a cross section of the Qinghai-Tibetan Plateau (QTP) from south (site A) to north (site K). The central diagram shows the hypothetical distribution of ecologically specialised taxa along the cross section.

Explicit environmental predictions and distribution of taxa allow the development of hypotheses on the biogeographic origin of biota and their trait and habitat evolution that can be studied in a classic hypothesis-testing framework using phylogenetic reconstructions and comparative methods or using model selection approaches (see Section III). The bottom diagrams show example predictions for scenarios of (1) ecological and trait diversification or stasis in a clade, and (2) biogeographic origin of clades.

To compare taxa, ecological stasis or diversification can be linked to phylogenetic biome conservatism ( sensu Crisp et al., ) or biome shifts. Within distinct clades diversification can be linked to patterns of phylogenetic niche conservatism ( sensu Crisp & Cook, ) with constraints on trait evolution or evolution of key innovations and/or adaptive diversification of traits (see methods in Section III.3). Specifically, on the basis of a dated multi-locus phylogeny, analysis of prevailing habitat preferences and character evolution provide a framework for testing if mountain and alpine species descended from alpine ancestors (phylogeny I: ecological stasis) or if they evolved from lowland ancestors via in-situ adaptation in novel azonal alpine habitats following the rise of the QTP (phylogeny II: ecological diversification). Analysis of phylogenetic relatedness among taxa can further reveal if the underlying processes occurred repeatedly. Ancestral area reconstructions (see methods in Section III.2) based on a multi-locus molecular phylogeny focussing on the regionally endemic alpine clades and carefully selected outgroups provide an independent test on the origin of the endemic alpine radiations of the QTP region.

Repeated long-distance colonisation will lead to unrelated taxa occupying neighbouring biomes or ecological habitats (phylogeny III). Vicariance following the rise of a mountain range can lead to distribution of sister taxa north and south of the range (phylogeny IV). Most of the studies available to date associate the diversification of organisms on the QTP with Middle to Late Miocene uplift and simultaneous climate changes, for example in lizards (Guo & Wang,; Guo et al., ), frogs (Che et al., ), butterflies (Leneveu, Chichvarkhin & Wahlberg, ), birds (Johansson et al.,; P ckert et al., ), and plants (Wang et al.,,; Favre et al.,; Jabbour & Renner,; Barres et al.,; Gao et al.,; Zhou et al.,; for a review see Wen et al., ), or with corresponding changes in drainage systems, for example in freshwater fishes (e.g. He & Chen,; Kang et al., ) and freshwater crabs (e.g. In the light of recent evidence, a significant part of the uplift of the QTP probably largely pre-dated the Miocene (Mulch & Chamberlain,, see below).

If mountain building did result in increased diversification rates, the lack of evidence for an earlier effect of the uplift on diversification might result from a natural delay of biological responses to environmental change, or from a warmer global climate that could have prevented a strong altitudinal zonation before and during the Middle Miocene climatic optimum (Fig. The choice of study groups biased towards younger taxa or methodological difficulties (e.g. The lack of fossilised records) might have further obscured the patterns of diversification. Additionally, studies on organismic evolution often do not offer a global perspective on biogeographic relationships between the QTP and other hotspots of biodiversity (a high proportion of taxonomic groups studied are endemics). It is clear, however, that biotic interchange contributed to the establishment of biodiversity hotspots, for example in the Andes (Cody et al., ) or on either side of Wallace's Line (Lohman et al.,; Richardson, Costion & Muellner, ).

The role of immigration is often overlooked in studies on the QTP, which renders the estimation of the relative importance of in-situ diversification versus immigration followed by allopatric speciation impossible. Finally, to date, there is no consensus scenario on geological and climatic changes in the region of the QTP against which evolutionary scientists could test their hypotheses.

Hence, this review aims to ( i) provide such a geological and palaeoclimatic scenario to promote hypothesis-based research, ( ii) highlight available methods to test for possible correlations between organismic diversification versus geological and climatic events during the formation of the QTP, and ( iii) review current knowledge on organismic evolution in the region of the QTP.