Immigration, species radiation and extinction in a highly diverse songbird lineage: white-eyes on Indian Ocean islands

Molecular phylogenetic hypotheses of species-rich lineages in regions where geological history can be reliably inferred may provide insights into the scale of processes driving diversification. Here we sample all extant or recently extinct white-eye (Zosterops) taxa of the southwest Indian Ocean, combined with samples from all principal continental lineages. Results support a high dispersal capability, with at least two independent continental sources for white-eyes of the region. An early (within 1.8 million years ago) expansion into the Indian Ocean may have originated either from Asia or Africa; the three resulting lineages show a disparate distribution consistent with considerable extinction following their arrival. Africa is supported as the origin of a later expansion into the region (within 1.2 million years ago). On two islands, a pair of Zosterops species derived from independent immigrations into the Indian Ocean co-occur or may have formerly co-occurred, providing strong support for their origin by double-island colonization rather than within-island (sympatric or microallopatric) speciation. On Mauritius and La Reunion, phylogenetic placement of sympatric white-eyes allow us to rule out a scenario in which independent within-island speciation occurred on both islands; one of the species pairs must have arisen by double colonization, while the other pair is likely to have arisen by the same mechanism. Long-distance immigration therefore appears to be responsible for much of the region's white-eye diversity. Independent immigrations into the region have resulted in lineages with mutually exclusive distributions and it seems likely that competition with congeneric species, rather than arrival frequency, may limit present-day diversity.

Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K+ channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A in order to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K+ homeostasis.

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.

NG2 cells differentiate into astrocytes in cerebellar slices

NG2-glia are an abundant population of glial cells that have been considered by many to be oligodendrocyte progenitor cells (OPCs). However, growing evidence suggests that NG2-glia may also be capable of differentiating into astrocytes and neurons under certain conditions. Here, we have examined NG2-glia in cerebellar slices, using transgenic mice in which the astroglial marker glial specific protein (GFAP) drives expression of the reporter gene enhanced green fluorescent protein (EGFP). Immunolabelling for NG2 shows that NG2-glia and GFAP-EGFP astroglia are separate populations in most areas of the brain, although a substantial population of NG2-glia in the pons also express the GFAP-EGFP reporter. In the cerebellum, NG2-glia did not express EGFP, either at postnatal day (P)12 or P29-30. We developed an organotypic culture of P12 cerebellar slices that maintain cytoarchitectural integrity of Purkinje neurons and Bergmann glia. In these cultures, BrdU labelling indicates that the majority of NG2-glia enter the cell cycle within 2 days in vitro (DIV), suggesting that NG2-glia undergo a [`]reactive' response in cerebellar cultures. After 2 DIV NG2-glia began to express the astroglial reporter EGFP and in some cases the respective GFAP protein. However, NG2-glia did not acquire phenotypic markers of neural stem cells or neurons. The results suggest that NG2-glia are not lineage restricted OPCs and are a potential source of astrocytes in the cerebellum.

Nitrosospira spp. can produce nitrous oxide via a nitrifier denitrification pathway

Nitrous oxide (N2O) emission from soils is a major contributor to the atmospheric loading of this potent greenhouse gas. It is thought that autotrophic ammonia oxidizing bacteria (AOB) are a significant source of soil-derived N2O and a denitrification pathway (i.e. reduction of NO2- to NO and N2O), so-called nitrifier denitrification, has been demonstrated as a N2O production mechanism in Nitrosomonas europaea. It is thought that Nitrosospira spp. are the dominant AOB in soil, but little information is available on their ability to produce N2O or on the existence of a nitrifier denitrification pathway in this lineage. This study aims to characterize N2O production and nitrifier denitrification in seven strains of AOB representative of clusters 0, 2 and 3 in the cultured Nitrosospira lineage. Nitrosomonas europaea ATCC 19718 and ATCC 25978 were analysed for comparison. The aerobically incubated test strains produced significant (P < 0.001) amounts of N2O and total N2O production rates ranged from 2.0 amol cell(-1) h(-1), in Nitrosospira tenuis strain NV12, to 58.0 amol cell(-1) h(-1), in N. europaea ATCC 19718. Nitrosomonas europaea ATCC 19718 was atypical in that it produced four times more N2O than the next highest producing strain. All AOB tested were able to carry out nitrifier denitrification under aerobic conditions, as determined by production of N-15-N2O from applied N-15-NO2-. Up to 13.5% of the N2O produced was derived from the exogenously applied N-15-NO2-. The results suggest that nitrifier denitrification could be a universal trait in the betaproteobacterial AOB and its potential ecological significance is discussed.

Molecular genetic differentiation in earthworms inhabiting a heterogeneous Pb-polluted landscape

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages ('A' and 'B'), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central 'ecological island' site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in ESTPb libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies.

Metal bioaccumulation and cellular fractionation in an epigeic earthworm (Lumbricus rubellus): the interactive influences of population exposure histories, site-specific geochemistry and mitochondrial genotype

Subcellular fractionation techniques were used to describe temporal changes (at intervals from T0 to T70 days) in the Pb, Zn and P partitioning profiles of Lumbricus rubellus populations from one calcareous (MDH) and one acidic (MCS) geographically isolated Pb/Zn-mine sites and one reference site (CPF). MDH and MCS individuals were laboratory maintained on their native field soils; CPF worms were exposed to both MDH and MCS soils. Site-specific differences in metal partitioning were found: notably, the putatively metal-adapted populations, MDH and MCS, preferentially partitioned higher proportions of their accumulated tissue metal burdens into insoluble CaPO4-rich organelles compared with naive counterparts, CPF. Thus, it is plausible that efficient metal immobilization is a phenotypic trait characterising metal tolerant ecotypes. Mitochondrial cytochrome oxidase II (COII) genotyping revealed that the populations indigenous to mine and reference soils belong to distinct genetic lineages, differentiated by 13%, with 7 haplotypes within the reference site lineage but fewer (3 and 4, respectively) in the lineage common to the two mine sites. Collectively, these observations raise the possibility that site-related genotype differences could influence the toxico-availability of metals and, thus, represent a potential confounding variable in field-based eco-toxicological assessments.

Genotypic sex determination enabled adaptive radiations of extinct marine reptiles

Adaptive radiations often follow the evolution of key traits, such as the origin of the amniotic egg and the subsequent radiation of terrestrial vertebrates. The mechanism by which a species determines the sex of its offspring has been linked to critical ecological and life-history traits(1-3) but not to major adaptive radiations, in part because sex-determining mechanisms do not fossilize. Here we establish a previously unknown coevolutionary relationship in 94 amniote species between sex-determining mechanism and whether a species bears live young or lays eggs. We use that relationship to predict the sex-determining mechanism in three independent lineages of extinct Mesozoic marine reptiles (mosasaurs, sauropterygians and ichthyosaurs), each of which is known from fossils to have evolved live birth(4-7). Our results indicate that each lineage evolved genotypic sex determination before acquiring live birth. This enabled their pelagic radiations, where the relatively stable temperatures of the open ocean constrain temperature-dependent sex determination in amniote species. Freed from the need to move and nest on land(4,5,8), extreme physical adaptations to a pelagic lifestyle evolved in each group, such as the fluked tails, dorsal fins and wing-shaped limbs of ichthyosaurs. With the inclusion of ichthyosaurs, mosasaurs and sauropterygians, genotypic sex determination is present in all known fully pelagic amniote groups (sea snakes, sirenians and cetaceans), suggesting that this mode of sex determination and the subsequent evolution of live birth are key traits required for marine adaptive radiations in amniote lineages.

The origin, molecular regulation and therapeutic potential of myogenic stem cell populations

Satellite cells, originating in the embryonic dermamyotome, reside beneath the myofibre of mature adult skeletal muscle and constitute the tissue-specific stem cell population. Recent advances following the identification of markers for these cells (including Pax7, Myf5, c-Met and CD34) (CD, cluster of differentiation; c-Met, mesenchymal epithelial transition factor) have led to a greater understanding of the role played by satellite cells in the regeneration of new skeletal muscle during growth and following injury. In response to muscle damage, satellite cells harbour the ability both to form myogenic precursors and to self-renew to repopulate the stem cell niche following myofibre damage. More recently, other stem cell populations including bone marrow stem cells, skeletal muscle side population cells and mesoangioblasts have also been shown to have myogenic potential in culture, and to be able to form skeletal muscle myofibres in vivo and engraft into the satellite cell niche. These cell types, along with satellite cells, have shown potential when used as a therapy for skeletal muscle wasting disorders where the intrinsic stem cell population is genetically unable to repair non-functioning muscle tissue. Accurate understanding of the mechanisms controlling satellite cell lineage progression and self-renewal as well as the recruitment of other stem cell types towards the myogenic lineage is crucial if we are to exploit the power of these cells in combating myopathic conditions. Here we highlight the origin, molecular regulation and therapeutic potential of all the major cell types capable of undergoing myogenic differentiation and discuss their potential therapeutic application.

Extreme host plant conservatism during at least 20 million years of host plant pursuit by oak gallwasps

Diversification of insect herbivores is often associated with coevolution between plant toxins and insect countermeasures, resulting in a specificity that restricts host plant shifts. Gall inducers, however, bypass plant toxins and the factors influencing host plant associations in these specialized herbivores remain unclear. We reconstructed the evolution of host plant associations in Western Palaearctic oak gallwasps (Cynipidae: Cynipini), a species-rich lineage of specialist herbivores on oak (Quercus). (1) Bayesian analyses of sequence data for three genes revealed extreme host plant conservatism, with inferred shifts between major oak lineages (sections Cerris and Quercus) closely matching the minimum required to explain observed diversity. It thus appears that the coevolutionary demands of gall induction constrain host plant shifts, both in cases of mutualism (e.g., fig wasps, yucca moths) and parasitism (oak gallwasps). (2) Shifts between oak sections occurred independently in sexual and asexual generations of the gallwasp lifecycle, implying that these can evolve independently. (3) Western Palaearctic gallwasps associated with sections Cerris and Quercus diverged at least 20 million years ago (mya), prior to the arrival of oaks in the Western Palaearctic from Asia 5-7 mya. This implies an Asian origin for Western Palaearctic gallwasps, with independent westwards range expansion by multiple lineages.

Cyclamen: time, sea and speciation biogeography using a temporally calibrated phylogeny

Aim The Mediterranean region is a species-rich area with a complex geographical history. Geographical barriers have been removed and restored due to sea level changes and local climatic change. Such barriers have been proposed as a plausible mechanism driving the high levels of speciation and endemism in the Mediterranean basin. This raises the fundamental question: is allopatric isolation the mechanism by which speciation occurs? This study explores the potential driving influence of palaeo-geographical events on the speciation of Cyclamen (Myrsinaceae), a group with most species endemic to the Mediterranean region. Cyclamen species have been shown experimentally to have few genetic barriers to hybridization. Location The Mediterranean region, including northern Africa, extending eastwards to the Black Sea coast. Methods A generic level molecular phylogeny of Myrsinaceae and Primulaceae is constructed, using Bayesian approximation, to produce a secondary age estimate for the stem lineage of Cyclamen. This estimate is used to calibrate temporally an infrageneric phylogeny of Cyclamen, built with nrDNA ITS, cpDNA trnL-F and cpDNA rps16 sequences. A biogeographical analysis of Cyclamen is performed using dispersal-vicariance analysis. Results The emergence of the Cyclamen stem lineage is estimated at 30.1-29.2 Ma, and the crown divergence at 12.9-12.2 Ma. The average age of Cyclamen species is 3.7 Myr. Every pair of sister species have mutually exclusive, allopatric distributions relative to each other. This pattern appears typical of divergence events throughout the evolutionary history of the genus. Main conclusions Geographical barriers, such as the varying levels of the Mediterranean Sea, are the most plausible explanation for speciation events throughout the phylogenetic history of Cyclamen. The genus demonstrates distributional patterns congruent with the temporally reticulate palaeogeography of the Mediterranean region.

Expression of FoxC, FoxF, FoxL1, and FoxQ1 genes in the dogfish scyliorhinus canicula defines ancient and derived roles for fox genes in vertebrate development

In the human genome, members of the FoxC, FoxF, FoxL1, and FoxQ1 gene families are found in two paralagous clusters. Here we characterize all four gene families in the dogfish Seyliorhinus canicula, a member of the cartilaginous fish lineage that diverged before the radiation of osteichthyan vertebrates. We identify two FoxC genes, two FoxF genes, and single FoxQ1 and FoxL1 genes, demonstrating cluster duplication preceded the radiation of gnathostomes. The expression of all six genes was analyzed by in situ hybridization. The results show conserved expression of FoxL1, FoxF, and FoxC genes in different compartments of the mesoderm and of FoxQ1 in pharyngeal endoderm and its derivatives, confirming these as ancient sites of Fox gene expression, and also illustrate multiple cases of lineage-specific expression domains. Comparison to invertebrate chordates shows that the majority of conserved vertebrate expression domains mark tissues that are part of the primitive chordate body plan.

The Fox genes of Branchiostoma floridae

The Fox genes are united by encoding a fork head domain, a deoxyribonucleic acid (DNA)-binding domain of the winged-helix type that marks these genes as encoding transcription factors. Vertebrate Fox genes are classified into 23 subclasses named from FoxA to FoxS. We have surveyed the genome of the amphioxus Branchiostoma floridae, identifying 32 distinct Fox genes representing 21 of these 23 subclasses. The missing subclasses, FoxR and FoxS, are specific to vertebrates, and in addition, B. floridae has one further group, FoxAB, that is not found in vertebrates. Hence, we conclude B. floridae has maintained a high level of Fox gene diversity. Expressed sequence tag and complementary DNA sequence data support the expression of 23 genes. Several linkages between B. floridae Fox genes were noted, including some that have evolved relatively recently via tandem duplication in the amphioxus lineage and others that are more ancient.

Evolutionary relationships of "candidatus riesia spp.," endosymbiotic enterobacteriaceae living within hematophagous primate lice

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse Pediculus humanus and provide new characterizations of the P-endosymbionts from Pediculus schaeffi from chimpanzees and Pthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium "Candidatus Arsenophonus insecticola." We propose that two additional species be added to the genus "Candidatus Riesia." The new species proposed within "Candidatus Riesia" have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for Buchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.