Inferring phylogenies from RAD sequence data

Reduced-representation genome sequencing represents a new source of data for systematics, and its potential utility in interspecific phylogeny reconstruction has not yet been explored. One approach that seems especially promising is the use of inexpensive short-read technologies (e.g., Illumina, SOLiD) to sequence restriction-site associated DNA (RAD)--the regions of the genome that flank the recognition sites of restriction enzymes. In this study, we simulated the collection of RAD sequences from sequenced genomes of different taxa (Drosophila, mammals, and yeasts) and developed a proof-of-concept workflow to test whether informative data could be extracted and used to accurately reconstruct "known" phylogenies of species within each group. The workflow consists of three basic steps: first, sequences are clustered by similarity to estimate orthology; second, clusters are filtered by taxonomic coverage; and third, they are aligned and concatenated for "total evidence" phylogenetic analysis. We evaluated the performance of clustering and filtering parameters by comparing the resulting topologies with well-supported reference trees and we were able to identify conditions under which the reference tree was inferred with high support. For Drosophila, whole genome alignments allowed us to directly evaluate which parameters most consistently recovered orthologous sequences. For the parameter ranges explored, we recovered the best results at the low ends of sequence similarity and taxonomic representation of loci; these generated the largest supermatrices with the highest proportion of missing data. Applications of the method to mammals and yeasts were less successful, which we suggest may be due partly to their much deeper evolutionary divergence times compared to Drosophila (crown ages of approximately 100 and 300 versus 60 Mya, respectively). RAD sequences thus appear to hold promise for reconstructing phylogenetic relationships in younger clades in which sufficient numbers of orthologous restriction sites are retained across species.     

A Veritable Menagerie of Heritable Bacteria from Ants,Butterflies, and Beyond: Broad Molecular Surveys and a Systematic Review

Maternally transmitted bacteria have been important players in the evolution of insects and other arthropods, affecting their nutrition, defense, development, and reproduction. Wolbachia are the best studied among these and typically the most prevalent. While several other bacteria have independently evolved a heritable lifestyle, less is known about their host ranges. Moreover, most groups of insects have not had their heritable microflora systematically surveyed across a broad range of their taxonomic diversity. To help remedy these shortcomings we used diagnostic PCR to screen for five groups of heritable symbionts—Arsenophonus spp., Cardinium hertigii, Hamiltonella defensa, Spiroplasma spp., and Wolbachia spp.—across the ants and lepidopterans (focusing, in the latter case, on two butterfly families—the Lycaenidae and Nymphalidae). We did not detect Cardinium or Hamiltonella in any host. Wolbachia were the most widespread, while Spiroplasma (ants and lepidopterans) and Arsenophonus (ants only) were present at low levels. Co-infections with different Wolbachia strains appeared especially common in ants and less so in lepidopterans. While no additional facultative heritable symbionts were found among ants using universal bacterial primers, microbes related to heritable enteric bacteria were detected in several hosts. In summary, our findings show that Wolbachia are the dominant heritable symbionts of ants and at least some lepidopterans. However, a systematic review of symbiont frequencies across host taxa revealed that this is not always the case across other arthropods. Furthermore, comparisons of symbiont frequencies revealed that the prevalence of Wolbachia and other heritable symbionts varies substantially across lower-level arthropod taxa. We discuss the correlates, potential causes, and implications of these patterns, providing hypotheses on host attributes that may shape the distributions of these influential bacteria.     

Kinetics of ADP dissociation from the trail and lead heads of actomyosin V following the power stroke

Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. (2006) Biochemistry 45, 13035-13045). The kinetics of ADP and deac-aminoADP dissociation from actomyosin V-HMM, following the power stroke, were determined using double-mixing stopped-flow fluorescence. These used either deac-aminoATP as the substrate with ADP or ATP chase or alternatively ATP as the substrate with either a deac-aminoADP or deac-aminoATP chase. Both sets of experiments show that the observed rate of ADP or deac-aminoADP dissociation from the trail head of actomyosin V-HMM is the same as from actomyosin V-S1. The dissociation of ADP from the lead head is decreased by up to 250-fold.     

Identification of somatic chromosomal abnormalities in hypothalamic hamartoma tissue at the GLI3 locus

Hypothalamic hamartomas (HH) are rare, benign congenital tumors associated with intractable epilepsy. Most cases are sporadic and nonsyndromic. Approximately 5% of HH cases are associated with Pallister-Hall syndrome (PHS), which is caused by haploinsufficiency of GLI3. We have investigated the possibility that HH pathogenesis in sporadic cases is due to a somatic (tumor-only) mutation in GLI3. We isolated genomic DNA from peripheral blood and surgically resected HH tissue in 55 patients with sporadic HH and intractable epilepsy. A genome-wide screen for loss of heterozygosity (LOH) and chromosomal abnormalities was performed with parallel analysis of blood and HH tissue with Affymetrix 10K SNP microarrays. Additionally, resequencing and fine mapping with SNP genotyping were completed for the GLI3 gene with comparisons between peripheral blood and HH tissue pairs. By analyzing chromosomal copy-number data for paired samples on the Affymetrix 10K array, we identified a somatic chromosomal abnormality on chromosome 7p in one HH tissue sample. Resequencing of GLI3 did not identify causative germline mutations but did identify LOH within the GLI3 gene in the HH tissue samples of three patients. Further genotyping of 28 SNPs within and surrounding GLI3 identified five additional patients exhibiting LOH. Together, these data provide evidence that the development of chromosomal abnormalities within GLI3 is associated with the pathogenesis of HH lesions in sporadic, nonsyndromic patients with HH and intractable epilepsy. Chromosomal abnormalities including the GLI3 locus were seen in 8 of 55 (15%) of the resected HH tissue samples. These somatic mutations appear to be highly variable.     

Porcine Encephalomyocarditis Virus Persists in Pig Myocardium and Infects Human Myocardial Cells

Recent advances toward using pig tissues in human transplantation have made it necessary to determine the risk of transmitting zoonotic viruses from pigs to humans or vice versa. We investigated the suitability of the porcine encephalomyocarditis virus (EMCV) model for such studies by determining its ability to persist in pigs, escape detection by routine serological methods, and infect human cells. Intraperitoneal inoculation of 5-week-old pigs with EMCV-30, a strain isolated from commercial pigs, resulted in acute cellular degeneration, infiltration of lymphocytes, and apoptosis in myocardium in 13 of 15 (86.7%) pigs during the acute phase of disease (3 to 21 days postinfection), followed by less-severe lymphocytic infiltration and apoptosis in 5 of 10 (50%) pigs during the chronic phase of the disease (day 45 to 90 postinfection). In the brain, lymphocytic infiltration, neuronal degeneration, and gliosis were observed in 26 to 33% of pigs in the acute phase of disease whereas perivascular cuffing was the predominant feature during chronic disease. EMCV antigens and RNA were demonstrated in the myocardium and brain during the chronic phase of disease. Analysis of 100 commercial pigs that were negative for EMCV antibodies identified two pig hearts positive for EMCV RNA. Porcine EMCV productively infected primary human cardiomyocytes as demonstrated by immunostaining using a monoclonal antibody specific for EMCV RNA polymerase, which is expressed only in productively infected cells, and by a one-step growth curve that showed production of 100 to 1,000 PFU of virus per cell within 6 h. The findings that porcine EMCV can persist in pig myocardium and can infect human myocardial cells make it an important infectious agent to screen for in pig-to-human cardiac transplants and a good model for xenozoonosis.     

The Role of Mouse Adenovirus Type 1 Early Region 1A in Acute and Persistent Infections in Mice

Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 10⁵ PFU of virus, an E1A null mutant, pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant, pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU, pmE109 and pmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.     

Defensive traits exhibit an evolutionary trade‐off and drive diversification in ants

Evolutionary biologists have long predicted that evolutionary trade‐offs among traits should constrain morphological divergence and species diversification. However, this prediction has yet to be tested in a broad evolutionary context in many diverse clades, including ants. Here, we reconstruct an expanded ant phylogeny representing 82% of ant genera, compile a new family‐wide trait database, and conduct various trait‐based analyses to show that defensive traits in ants do exhibit an evolutionary trade‐off. In particular, the use of a functional sting negatively correlates with a suite of other defensive traits including spines, large eye size, and large colony size. Furthermore, we find that several of the defensive traits that trade off with a sting are also positively correlated with each other and drive increased diversification, further suggesting that these traits form a defensive suite. Our results support the hypothesis that trade‐offs in defensive traits significantly constrain trait evolution and influence species diversification in ants.     

Diversity of rodent and shrew assemblages in different vegetation types of the savannah biome in South Africa: no evidence for nested subsets or competition

Identifying nonrandom species composition patterns predicted by assembly rules has been a central theme in community ecology. Few studies have investigated the prevalence of multiple drivers on species composition patterns in small mammal assemblages in the Old World. This study investigated seasonal changes in rodent and shrew diversity in eleven savannah vegetation types in South Africa. We tested whether species composition patterns are nonrandom with respect to predictions from Diamond's assembly rules, niche limitation hypothesis and nestedness hypothesis. Species richness estimators indicated that inventories for the rodents (80%) and shrews (100%) were relatively complete. Rodent (n = 11 species) diversity and shrew (n = 5 species) diversity were highest in summer and lowest in autumn. Rodent richness was highest in the Terminalia sericea bushveld and woodlands and lowest in the Drypetes arguta sand forest, whilst shrew richness was highest in the T. sericea bushveld and woodlands and lowest in the Acacia nilotica/Dichrostachys cinerea open shrub savannah. We found no support for the predictions of competition and nestedness hypotheses and suggest that this was probably due to the high seasonal and annual variability in rodent and shrew diversity.