Repeated Reticulate Evolution in North American Papilio machaon Group Swallowtail Butterflies

Hybridization between distinct populations or species is increasingly recognized as an important process for generating biodiversity. However, the interaction between hybridization and speciation is complex, and the diverse evolutionary outcomes of hybridization are difficult to differentiate. Here we characterize potential hybridization in a species group of swallowtail butterflies using microsatellites, DNA sequences, and morphology, and assess whether adaptive introgression or homoploid hybrid speciation was the primary process leading to each putative hybrid lineage. Four geographically separated hybrid populations were identified in the Papilio machaon species group. One distinct mitochondrial DNA clade from P. machaon was fixed in three hybrid taxa (P. brevicauda, P. joanae, and P. m. kahli), while one hybrid swarm (P. zelicaon x machaon) exhibited this hybrid mtDNA clade as well as widespread parental mtDNA haplotypes from both parental species. Microsatellite markers and morphology showed variable admixture and intermediacy, ranging from signatures of prolonged differential introgression from the paternal species (P. polyxenes/P. zelicaon) to current gene flow with both parental species. Divergences of the hybrid lineages dated to early- to mid-Pleistocene, suggesting that repeated glaciations and subsequent range shifts of parental species, particularly P. machaon hudsonianus, facilitated initial hybridization. Although each lineage is distinct, P. joanae is the only taxon with sufficient evidence (ecological separation from parental species) to define it as a homoploid hybrid species. The repetition of hybridization in this group provides a valuable foundation for future research on hybridization, and these results emphasize the potential for hybridization to drive speciation in diverse ways.     

Inwardly rectifying K(+) channels in spermatogenic cells: functional expression and implication in sperm capacitation

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K(+) channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K(+)-selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K(+) and its magnitude was dependent on the external K(+) concentration. The channels selected K(+) over other monovalent cations. Current was virtually absent when external K(+) was replaced with Na(+) or N-methyl-D-glucamine. Addition of Cs(+) or Ba(2+) (IC(50) of approximately 15 microM) to the external solution effectively blocked K(+) current. Dialyzing the cells with a Mg(2+)-free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from -52 +/- 6 to -66 +/- 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba(2+) during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K(+) channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.     

Role of Caribbean Islands in the diversification and biogeography of Neotropical Heraclides swallowtails

Numerous hypotheses on the evolution of Neotropical biodiversity have stimulated research to provide a better understanding of diversity dynamics and distribution patterns of the region. However, few studies integrate molecular and morphological data with complete sampling of a Neotropical group, and so there has been little synthesis of the multiple processes governing biodiversity through space and time. Here, a total‐evidence phylogenetic approach is used to reconstruct the evolutionary history of the butterfly subgenus Heraclides. We used DNA sequences for two mitochondrial genes and one nuclear gene and coded 133 morphological characters of larvae and adults. A robust and well‐resolved phylogeny was obtained using several analytical approaches, while molecular dating and biogeographical analyses indicated an early Miocene origin (22 Mya) in the Caribbean Islands. We inferred six independent dispersal events from the Caribbean to the mainland, and three from the mainland to the Caribbean, and we suggest that cooling climates with decreasing sea levels may have contributed to these events. The time‐calibrated tree is best explained by a museum model of diversity in which both speciation and extinction rates remained constant through time. By assessing both continental and fine‐scale biodiversity patterns, this study provides new findings, for instance that islands may act as source of diversity rather than as a sink, to explain spatio‐temporal macroevolutionary processes within the Neotropical region.     

Deciphering the ovarian cancer ascites fluid peptidome

Background

Conventional proteomic approaches have thus far been unable to identify novel serum biomarkers for ovarian cancer that are more sensitive and specific than the current clinically used marker, CA-125. Because endogenous peptides are smaller and may enter the circulation more easily than proteins, a focus on the low-molecular-weight region may reveal novel biomarkers with enhanced sensitivity and specificity. In this study, we deciphered the peptidome of ascites fluid from 3 ovarian cancer patients and 3 benign individuals (ascites fluid from patients with liver cirrhosis).

Results

Following ultrafiltration of the ascites fluids to remove larger proteins, each filtrate was subjected to solid phase extraction and fractionated using strong cation exchange chromatography. The resultant fractions were analyzed using an Orbitrap mass spectrometer. We identified over 2000 unique endogenous peptides derived from 259 proteins. We then catalogued over 777 peptides that were found only in ovarian cancer ascites. Our list of peptides found in ovarian cancer specimens includes fragments derived from the proteins vitronectin, transketolase and haptoglobin.

Conclusions

Peptidomics may uncover previously undiscovered disease-specific endogenous peptides that warrant further investigation as biomarkers for ovarian cancer.     

Characterization of Clostridium thermocellum Isolates Grown on Cellulose and Sugarcane Bagasse

Although plant cell walls may be degraded by microbial free enzymes, many bacteria degrade cellulose via enzyme complexes called cellulosomes. The study of the structures and mechanisms of these large macromolecular complexes is an active and ongoing research topic, with the goal of developing methods to improve lignocellulosic biomass conversion using cellulosomes. The aim of the present work was to evaluate and characterize the holocellulolytic activities produced by two new isolates (ISO1 and ISO2) of the spore-forming thermophilic anaerobic bacterium Clostridium thermocellum, during growth on crystalline cellulose and sugarcane bagasse, in comparison with activities obtained from the C. thermocellum strain CthJW. The pH and temperature values for optimal growth of the isolates were pH 7 and 60 °C, respectively. The isolates produced cellulolytic, xylanolytic, and pectinolytic activities when cultured on crystalline cellulose or sugarcane bagasse, which have never been used previously as the sole carbon source for these bacteria. The profiles of secreted proteins for these isolates, ISO1 and ISO2, were quite different from those obtained for the standard strain CthJW and from each other, as shown by 2D gel electrophoresis maps, and these profiles also depend on the carbon source used. Different protein isoforms were also detected in the maps for all growth conditions and bacterial strains. MALDI-TOF mass spectrometry was used to identify the differentially expressed proteins for ISO1 and ISO2 under growth in the presence of cellulose as carbon source. Twenty-five differentially expressed spots were identified and grouped into 8 functional categories: metabolism (20 %), motor function (20 %), protein synthesis (12 %), oxidative stress (16 %), secretory pathway (12 %), cellulose hydrolysis (4 %), protein folding (4 %), and defense (12 %). Spots 200 and 197, identified as a glycosyl hydrolase family member 9 and as a chaperone GroEL, respectively, were detected for all isolates and are potentially related to cellulosome architecture.     

Monitoring the stability of crosslinked protein crystals biotemplates: a feasibility study

Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein molecules in the crystal is a complementary 3D array of voids made of interconnected cavities and exhibiting highly ordered porosity. The permeability of the porosity of chemically crosslinked enzyme protein crystals to low molecular weight solutes, was used for enzyme mediated organic synthesis and size exclusion chromatography. This permeability might be extended to explore new potential applications for protein crystals, for example, their use as bio-templates for the fabrication of novel, nano-structured composite materials. The quality of composites obtained from "filling" of the ordered voids in protein crystals and their potential applications will be strongly dependent upon an accurate preservation of the order in the original protein crystal 3D array during the "filling" process. Here we propose and demonstrate the feasibility of monitoring the changes in 3D order of the protein array by a step-by-step molecular level monitoring of a model system for hydrogel bio-templating by glutaraldehyde crosslinked lysozyme crystals. This monitoring is based on step-by-step comparative analysis of data obtained from (i) X-ray crystallography: resolution, unit cell dimensions and B-factor values and (ii) fluorescence decay kinetics of ultra-fast laser activated dye, impregnated within these crystals. Our results demonstrated feasibility of the proposed monitoring approach and confirmed that the stabilized protein crystal template retained its 3D structure throughout the process.     

Metabolite Profiling of Anti‐Addictive Alkaloids from Four Mexican Tabernaemontana Species and the Entheogenic African Shrub Tabernanthe iboga (Apocynaceae)

Ibogaine and other ibogan type alkaloids present anti‐addictive effects against several drugs of abuse and occur in different species of the Apocynaceae family. In this work, we used gas chromatography‐mass spectrometry (GC/MS) and principal component analysis (PCA) in order to compare the alkaloid profiles of the root and stem barks of four Mexican Tabernaemontana species with the root bark of the entheogenic African shrub Tabernanthe iboga. PCA demonstrated that separation between species could be attributed to quantitative differences of the major alkaloids, coronaridine, ibogamine, voacangine, and ibogaine. While T. iboga mainly presented high concentrations of ibogaine, Tabernaemontana samples either showed a predominance of voacangine and ibogaine, or coronaridine and ibogamine, respectively. The results illustrate the phytochemical proximity between both genera and confirm previous suggestions that Mexican Tabernaemontana species are viable sources of anti‐addictive compounds.     

Phylogenetic analysis of cuckoo wasps (Hymenoptera: Chrysididae) reveals a partially artificial classification at the genus level and a species‐rich clade of bee parasitoids

Cuckoo wasps (Hymenoptera: Chrysididae) are a species‐rich family of obligate brood parasites (i.e. parasitoids and kleptoparasites) whose hosts range from sawflies, wasps and bees, to walking sticks and moths. Their brood parasitic lifestyle has led to the evolution of fascinating adaptations, including chemical mimicry of host odours by some species. Long‐term nomenclatural stability of the higher taxonomic units (e.g. genera, tribes, and subfamilies) in this family and a thorough understanding of the family's evolutionary history critically depend on a robust phylogeny of cuckoo wasps. Here we present the results from phylogenetically analysing ten nuclear‐encoded genes and one mitochondrial gene, all protein‐coding, in a total of 186 different species of cuckoo wasps representing most major cuckoo wasp lineages. The compiled data matrix comprised 4946 coding nucleotide sites and was phylogenetically analysed using classical maximum‐likelihood and Bayesian inference methods. The results of our phylogenetic analyses are mostly consistent with earlier ideas on the phylogenetic relationships of the cuckoo wasps' subfamilies and tribes, but cast doubts on the hitherto hypothesized phylogenetic position of the subfamily Amiseginae. However, the molecular data are not fully conclusive in this respect due to low branch support values at deep nodes. In contrast, our phylogenetic estimates clearly indicate that the current systematics of cuckoo wasps at the genus level is artificial. Several of the currently recognized genera are para‐ or polyphyletic (e.g. Cephaloparnops, Chrysis, Chrysura, Euchroeus, Hedychridium, Praestochrysis, Pseudochrysis, Spintharina, and Spinolia). At the same time, our data support the validity of the genus Colpopyga, previously synonymized with Hedychridium. We discuss possible solutions for how to resolve the current shortcomings in the systematics of cuckoo wasp genera and decided to grant Prospinolia the status of a valid genus (Prospinolia stat.n. ) and transferring Spinolia theresae [du Buysson 1900] from Spinolia to Prospinolia (Prospinolia theresae stat.restit. ). We discuss the implications of our phylogenetic inferences for understanding the evolution of host associations in this group. The results of our study not only shed new light on the evolutionary history of cuckoo wasps, but also set the basis for future phylogenomic investigations on this captivating group of wasps by guiding taxonomic sampling efforts and the design of probes for target DNA enrichment approaches.