Membrane cholesterol modulates STEAP2 conformation during dynamic intracellular trafficking processes leading to broad subcellular distribution.

STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported. Leveraging a newly-identified monoclonal antibody that recognizes a conformation-sensitive epitope nested in the second extracellular loop of STEAP2, we demonstrate that the epitope formation was dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Monitoring the STEAP2-dependent internalization of this antibody uncovered STEAP2's rapid internalization from the cell surface and their subsequence trafficking to the Golgi region and endosome-like puncta. Acute inhibition of endocytosis also increased the detectable amount of STEAP2 at the plasma membrane. Collectively, these experiments demonstrate that an intricate balance of membrane flux between the secretory and endocytic pathways underlies the characteristic broad subcellular localization of STEAP2. By using a cell-based assay that detects the metalloreductase functions of cell surface-localizing STEAP4, STEAP2's metalloreductase activities were not detectable, suggesting that its enzymatic function is suppressed at the plasma membrane. The conformational modulation of STEAP2 by the local membrane cholesterol content can therefore serve as a potential mechanism to modulate STEAP2 function in a compartment-restricted manner, by coupling a pre-existing difference in cholesterol content among different cellular membranes to a dynamic trafficking process leading to broad subcellular distribution.

Improving phylogenetic inference of core Chlorophyta using chloroplast sequences with strong phylogenetic signals and heterogeneous models.

Phylogenetic relationships within the green algal phylum Chlorophyta have proven difficult to resolve. The core Chlorophyta include Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Pedinophyceae and Chlorodendrophyceae, but the relationships among these classes remain unresolved and the monophyly of Ulvophyceae and Trebouxiophyceae are highly controversial. We analyzed a dataset of 101 green algal species and 73 protein-coding genes sampled from complete and partial chloroplast genomes, including six newly sequenced ulvophyte genomes (Blidingia minima NIES-1837, Ulothrix zonata, Halochlorococcum sp. NIES-1838, Scotinosphaera sp. NIES-154, Caulerpa brownii and Cephaleuros sp. HZ-2017). We applied the Tree Certainty (TC) score to quantify the level of incongruence between phylogenetic trees in chloroplast genomic datasets, and show that the conflicting phylogenetic trees of core Chlorophyta stem from the most GC-heterogeneous sites. With removing the most GC-heterogeneous sites, our chloroplast phylogenomic analyses using heterogeneous models consistently support monophyly of the Chlorophyceae and of the Trebouxiophyceae, but the Ulvophyceae was resolved as polyphyletic. Our analytical framework provides an efficient approach to reconstruct the optimal phylogenetic relationships by minimizing conflicting signals.

Effects of Pressure and pH on the Hydrolysis of Cytosine: Implications for Nucleotide Stability around Deep-Sea Black Smokers.

The relatively low chemical stability of cytosine compared with other nucleobases is a key concern in origin-of-life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to uracil remains unknown. Through in situ NMR spectroscopy measurements, it has been determined that the half-life of cytosine at 373.15 K decreases from (18.0±0.7) days at ambient pressure (0.1 MPa) to (8.64±0.18) days at high pressure (200 MPa). This yields an activation volume for hydrolysis of (-11.8±0.5) cm3 mol-1 ; a decrease that is similar to the molar volume of water (18.0 cm3 mol-1 ) and consistent with a tetrahedral 3,3-hydroxyamine transition-state/intermediate species. Similar behaviour was also observed for cytidine. At both ambient and high pressures, the half-life of cytosine decreases significantly as the pH decreases from 7.0 to 6.0. These results provide scant support for the notion that RNA-based life forms originated in high-temperature, high-pressure, acidic environments.

Chloroplast Phylogenomic Inference of Green Algae Relationships.

The green algal phylum Chlorophyta has six diverse classes, but the phylogenetic relationship of the classes within Chlorophyta remains uncertain. In order to better understand the ancient Chlorophyta evolution, we have applied a site pattern sorting method to study compositional heterogeneity and the model fit in the green algal chloroplast genomic data. We show that the fastest-evolving sites are significantly correlated with among-site compositional heterogeneity, and these sites have a much poorer fit to the evolutionary model. Our phylogenomic analyses suggest that the class Chlorophyceae is a monophyletic group, and the classes Ulvophyceae, Trebouxiophyceae and Prasinophyceae are non-monophyletic groups. Our proposed phylogenetic tree of Chlorophyta will offer new insights to investigate ancient green algae evolution, and our analytical framework will provide a useful approach for evaluating and mitigating the potential errors of phylogenomic inferences.

Does the Ribosome Challenge our Understanding of the RNA World?

In a recent article published in these pages, Bowman and colleagues propose that the ribosome represents a challenge to the RNA world model, a long-standing framework to explain the origin of DNA and genetically encoded proteins from a hypothetical RNA-based system. Specifically, they outline a scenario for the emergence and subsequent coevolution of the peptidyl transferase centre (PTC) of the ribosome with non-templated peptide products of this RNA through chemical evolution. They also propose that the PTC would have predated the emergence of enzymatic RNA replication, and that this in turn indicates that the RNA world never existed. We and others have previously incorporated non-templated peptide production as an early stage in the evolution of protein synthesis, which we would count as a chemical process, in agreement with Bowman and colleagues' model. However, their model raises an important question: to what extent could early protein synthesis and its products have evolved in the absence of Darwinian processes? We argue that evolution of the early ribosome requires Darwinian evolution, and that, while chemical evolution could give rise to peptidyl transferase activity, it is insufficient for subsequent improvement of a proto-PTC, or for ongoing coevolution of the proto-PTC with its early non-templated peptide products. We conclude that it is difficult to preclude the involvement of replicative processes, themselves subject to Darwinian evolution, from the evolution of the PTC. Finally, Bowman et al. call into question current models for the RNA to protein transition. We show that the difficulty that Bowman et al. have with this scenario is down to a misreading of our previous work.

The complete mitochondrial genome of the eastern grey kangaroo (Macropus giganteus).

We present the complete mitochondrial genome (accession number: LK995454) of an iconic Australian species, the eastern grey kangaroo (Macropus giganteus). The mitogenomic organization is consistent with other marsupials, encoding 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, an origin of light strand replication and a control region or D-loop. No repetitive sequences were detected in the control region. The M. giganteus mitogenome exemplifies a combination of tRNA gene order and structural peculiarities that appear to be unique to marsupials. We present a maximum likelihood phylogeny based on complete mitochondrial protein and RNA coding sequences that confirms the phylogenetic position of the grey kangaroo among macropodids.

New Zealand Passerines Help Clarify the Diversification of Major Songbird Lineages during the Oligocene.

Passerines are the largest avian order, and the 6,000 species comprise more than half of all extant bird species. This successful radiation probably had its origin in the Australasian region, but dating this origin has been difficult due to a scarce fossil record and poor biogeographic assumptions. Many of New Zealand's endemic passerines fall within the deeper branches of the passerine radiation, and a well resolved phylogeny for the modern New Zealand element in the deeper branches of the oscine lineage will help us understand both oscine and passerine biogeography. To this end we present complete mitochondrial genomes representing all families of New Zealand passerines in a phylogenetic framework of over 100 passerine species. Dating analyses of this robust phylogeny suggest Passeriformes originated in the early Paleocene, with the major lineages of oscines "escaping" from Australasia about 30 Ma, and radiating throughout the world during the Oligocene. This independently derived conclusion is consistent with the passerine fossil record.

The Origin of Land Plants: A Phylogenomic Perspective.

Land plants are a natural group, and Charophyte algae are the closest lineages of land plants and have six morphologically diverged groups. The conjugating green algae (Zygnematales) are now suggested to be the extant sister group to land plants, providing the novel understanding for character evolution and early multicellular innovations in land plants. We review recent molecular phylogenetic work on the origin of land plants and discuss some future directions in phylogenomic analyses.

Epigenetics, Darwin, and Lamarck.

It is not really helpful to consider modern environmental epigenetics as neo-Lamarckian; and there is no evidence that Lamarck considered the idea original to himself. We must all keep learning about inheritance, but attributing modern ideas to early researchers is not helpful, and can be misleading.

Two fundamental questions about protein evolution.

Two basic questions are considered that approach protein evolution from different directions; the problems arising from using Markov models for the deeper divergences, and then the origin of proteins themselves. The real problem for the first question (going backwards in time) is that at deeper phylogenies the Markov models of sequence evolution must lose information exponentially at deeper divergences, and several testable methods are suggested that should help resolve these deeper divergences. For the second question (coming forwards in time) a problem is that most models for the origin of protein synthesis do not give a role for the very earliest stages of the process. From our knowledge of the importance of replication accuracy in limiting the length of a coding molecule, a testable hypothesis is proposed. The length of the code, the code itself, and tRNAs would all have prior roles in increasing the accuracy of RNA replication; thus proteins would have been formed only after the tRNAs and the length of the triplet code are already formed. Both questions lead to testable predictions.

Cooperation and selfishness both occur during molecular evolution.

Perhaps the 'selfish' aspect of evolution has been over-emphasised, and organisms considered as basically selfish. However, at the macromolecular level of genes and proteins the cooperative aspect of evolution is more obvious and balances this self-centred aspect. Thousands of proteins must function together in an integrated manner to use and to produce the many molecules necessary for a functioning cell. The macromolecules have no idea whether they are functioning cooperatively or competitively with other genes and gene products (such as proteins). The cell is a giant cooperative system of thousands of genes/proteins that function together, even if it has to simultaneously resist 'parasites'. There are extensive examples of cooperative behavior among genes and proteins in both functioning cells and in the origin of life, so this cooperative nature, along with selfishness, must be considered part of normal evolution. The principles also apply to very large numbers of examples of 'positive interactions' between organisms, including both eukaryotes and akaryotes (prokaryotes). This does not negate in any way the 'selfishness' of genes - but macromolecules have no idea when they are helping, or hindering, other groups of macromolecules. We need to assert more strongly that genes, and gene products, function together as a cooperative unit.

The relative ages of eukaryotes and akaryotes.

The Last Eukaryote Common Ancestor (LECA) appears to have the genetics required for meiosis, mitosis, nucleus and nuclear substructures, an exon/intron gene structure, spliceosomes, many centres of DNA replication, etc. (and including mitochondria). Most of these features are not generally explained by models for the origin of the Eukaryotic cell based on the fusion of an Archeon and a Bacterium. We find that the term 'prokaryote' is ambiguous and the non-phylogenetic term akaryote should be used in its place because we do not yet know the direction of evolution between eukaryotes and akaryotes. We use the term 'protoeukaryote' for the hypothetical stem group ancestral eukaryote that took up a bacterium as an endosymbiont that formed the mitochondrion. It is easier to make detailed models with a eukaryote to an akaryote transition, rather than vice versa. So we really are at a phylogenetic impasse in not being confident about the direction of change between eukaryotes and akaryotes.

Phylogenetic species identification in Rattus highlights rapid radiation and morphological similarity of New Guinean species.

The genus Rattus is highly speciose, the taxonomy is complex, and individuals are often difficult to identify to the species level. Previous studies have demonstrated the usefulness of phylogenetic approaches to identification in Rattus but some species, especially among the endemics of the New Guinean region, showed poor resolution. Possible reasons for this are simple misidentification, incomplete gene lineage sorting, hybridization, and phylogenetically distinct lineages that are unrecognised taxonomically. To assess these explanations we analysed 217 samples, representing nominally 25 Rattus species, collected in New Guinea, Asia, Australia and the Pacific. To reduce misidentification problems we sequenced museum specimens from earlier morphological studies and recently collected tissues from samples with associated voucher specimens. We also reassessed vouchers from previously sequenced specimens. We inferred combined and separate phylogenies from two mitochondrial DNA regions comprising 550 base pair D-loop sequences and both long (655 base pair) and short (150 base pair) cytochrome oxidase I sequences. Our phylogenetic species identification for 17 species was consistent with morphological designations and current taxonomy thus reinforcing the usefulness of this approach. We reduced misidentifications and consequently the number of polyphyletic species in our phylogenies but the New Guinean Rattus clades still exhibited considerable complexity. Only three of our eight New Guinean species were monophyletic. We found good evidence for either incomplete mitochondrial lineage sorting or hybridization between species within two pairs, R. leucopus/R. cf. verecundus and R. steini/R. praetor. Additionally, our results showed that R. praetor, R. niobe and R. verecundus each likely encompass more than one species. Our study clearly points to the need for a revised taxonomy of the rats of New Guinea, based on broader sampling and informed by both morphology and phylogenetics. The remaining taxonomic complexity highlights the recent and rapid radiation of Rattus in the Australo-Papuan region.

Two new fern chloroplasts and decelerated evolution linked to the long generation time in tree ferns.

We report the chloroplast genomes of a tree fern (Dicksonia squarrosa) and a "fern ally" (Tmesipteris elongata), and show that the phylogeny of early land plants is basically as expected, and the estimates of divergence time are largely unaffected after removing the fastest evolving sites. The tree fern shows the major reduction in the rate of evolution, and there has been a major slowdown in the rate of mutation in both families of tree ferns. We suggest that this is related to a generation time effect; if there is a long time period between generations, then this is probably incompatible with a high mutation rate because otherwise nearly every propagule would probably have several lethal mutations. This effect will be especially strong in organisms that have large numbers of cell divisions between generations. This shows the necessity of going beyond phylogeny and integrating its study with other properties of organisms.

Phylogenetic position of avian nocturnal and diurnal raptors.

We report three new avian mitochondrial genomes, two from widely separated groups of owls and a falcon relative (the Secretarybird). We then report additional progress in resolving Neoavian relationships in that the two groups of owls do come together (it is not just long-branch attraction), and the Secretarybird is the deepest divergence on the Accipitridae lineage. This is now agreed between mitochondrial and nuclear sequences. There is no evidence for the monophyly of the combined three groups of raptors (owls, eagles, and falcons), and again this is agreed by nuclear and mitochondrial sequences. All three groups (owls, accipitrids [eagles], and falcons) do appear to be members of the "higher land birds," and though there may not yet be full "consilience" between mitochondrial and nuclear sequences for the precise order of divergences of the eagles, falcons, and the owls, there is good progress on their relationships.

Streptophyte algae and the origin of land plants revisited using heterogeneous models with three new algal chloroplast genomes.

The phylogenetic branching order of the green algal groups that gave rise to land plants remains uncertain despite its fundamental importance to understanding plant evolution. Previous studies have demonstrated that land plants evolved from streptophyte algae, but different lineages of streptophytes have been suggested to be the sister group of land plants. To better understand the evolutionary history of land plants and to determine the potential effects of "long-branch attraction" in phylogenetic reconstruction, we analyzed a chloroplast genome data set including three new chloroplast genomes from streptophyte algae: Coleochaetae orbicularis (Coleochaetales), Nitella hookeri (Charales), and Spirogyra communis (Zygnematales). We further applied a site pattern sorting method together with site- and time-heterogeneous models to investigate the branching order among streptophytes and land plants. Our chloroplast phylogenomic analyses support previous hypotheses based on nuclear data in placing Zygnematales alone, or a clade consisting of Coleochaetales plus Zygnematales, as the closest living relatives of land plants.