On a limitation of Zeeman polarimetry and imperfect instrumentation in representing solar magnetic fields with weaker polarization signal.

Full disk vector magnetic fields are used widely for developing better understanding of large-scale structure, morphology, and patterns of the solar magnetic field. The data are also important for modeling various solar phenomena. However, observations of vector magnetic fields have one important limitation that may affect the determination of the true magnetic field orientation. This limitation stems from our ability to interpret the differing character of the Zeeman polarization signals which arise from the photospheric line-of-sight vs. the transverse components of the solar vector magnetic field, and is likely exacerbated by unresolved structure (non-unity fill fraction) as well as the disambiguation of the 180° degeneracy in the transverse-field azimuth. Here we provide a description of this phenomenon, and discuss issues, which require additional investigation.

Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand.

Resistance of Plasmodium falciparum to artemisinin combination therapy (ACT) in Southeast Asia can have a devastating impact on chemotherapy and control measures. In this study, the evolution of artemisinin-resistant P. falciparum in Thailand was assessed by exploring mutations in the K13 locus believed to confer drug resistance phenotype. P. falciparum-infected blood samples were obtained from patients in eight provinces of Thailand over two decades (1991-2014; n = 904). Analysis of the K13 gene was performed by either sequencing the complete coding region (n = 259) or mutation-specific PCR-restriction fragment length polymorphism method (n = 645). K13 mutations related to artesunate resistance were detected in isolates from Trat province bordering Cambodia in 1991, about 4 years preceding widespread deployment of ACT in Thailand and increased in frequency over time. Nonsynonymous nucleotide diversity exceeded synonymous nucleotide diversity in the propeller region of the K13 gene, supporting the hypothesis that this diversity was driven by natural selection. No single mutant appeared to be favoured in every population, and propeller-region mutants were rarely observed in linkage with each other in the same haplotype. On the other hand, there was a highly significant association between the occurrence of a propeller mutant and the insertion of two or three asparagines after residue 139 of K13. Whether this insertion plays a compensatory role for deleterious effects of propeller mutants on the function of the K13 protein requires further investigation. However, modification of duration of ACT from 2-day to 3-day regimens in 2008 throughout the country does not halt the increase in frequency of mutants conferring artemisinin resistance phenotype.

Evolution of adaptive phenotypic traits without positive Darwinian selection.

Recent evidence suggests the frequent occurrence of a simple non-Darwinian (but non-Lamarckian) model for the evolution of adaptive phenotypic traits, here entitled the plasticity-relaxation-mutation (PRM) mechanism. This mechanism involves ancestral phenotypic plasticity followed by specialization in one alternative environment and thus the permanent expression of one alternative phenotype. Once this specialization occurs, purifying selection on the molecular basis of other phenotypes is relaxed. Finally, mutations that permanently eliminate the pathways leading to alternative phenotypes can be fixed by genetic drift. Although the generality of the PRM mechanism is at present unknown, I discuss evidence for its widespread occurrence, including the prevalence of exaptations in evolution, evidence that phenotypic plasticity has preceded adaptation in a number of taxa and evidence that adaptive traits have resulted from loss of alternative developmental pathways. The PRM mechanism can easily explain cases of explosive adaptive radiation, as well as recently reported cases of apparent adaptive evolution over ecological time.

Reduced variance of gene expression at numerous loci in a population of chickens selected for high feather pecking.

Changes in gene expression in response to selection were studied by comparing microarray expression profiles among a population of domestic chickens selected for high feather pecking (FP) with a control population and a population selected for low FP. No transcripts showed significant differences among populations with respect to mean expression scores, but numerous transcripts showed reduced variance in expression scores in the high FP population in comparison to control and low FP populations. The reduction in variance in the high FP population generally involved transcripts whose expression scores had a negatively skewed distribution in the control population but not in the high FP population. A certain number of these transcripts corresponded to genes whose expression was significantly associated with either severe FP (SFP) or gentle FP. The patterns of gene expression associated with SFP and gentle FP were distinct, suggesting that very distinct underlying neural mechanisms underlie these 2 behaviors, with SFP showing more signs of an association with synaptic plasticity and with an immunosuppressive stress response.

Effect of Quantum phytase on nutrient digestibility and bone ash in White Leghorn laying hens fed corn-soybean meal-based diets.

The efficacy of an Escherichia coli 6-phytase supplementation (Quantum) on nutrient digestibility-retention and bone ash in laying hens fed corn-soybean meal (CSM) diets was investigated. White Leghorn hens (Shaver and Bovan strains) were fed CSM diets containing 0.35% (positive control, PC), 0.25% (negative control 1, NC1), or 0.15% (negative control 2, NC2) nonphytate P from 21 to 61 wk of age. Six more diets were manufactured by supplementing the negative control diets with 200, 400, and 600 units per kilogram of exogenous phytase resulting in a total of 9 treatments. Each dietary treatment x strain subclass was replicated twice with 6 hens per replication. Fecal and ileal digesta samples were collected at 42 wk of age to determine apparent nutrient digestibility or retention. Left tibiae were collected at 42 and 61 wk of age to determine bone ash. The coefficients for ileal digestibility and fecal retention for protein were higher (P < 0.05) for the unsupplemented negative control treatments compared with the PC. A linear reduction in phytate digestibility and ileal protein digestibility was reported with increasing levels of phytase to the NC1 diet. Phytase addition to the NC1 treatment resulted in a linear decrease in the digestibility of amino acids except for methionine and proline. Significantly higher phytate digestibility was demonstrated with the NC2 treatment containing 400 units per kilogram of phytase compared with the PC. Tibial bone ash percentage was higher (P < 0.05) in 61-wk-old hens fed 200 or 400 units per kilogram of phytase-supplemented NC2 diets. Significantly higher diet AME and fecal protein retention were demonstrated for Shaver hens in comparison to the Bovan hens. Overall, the Quantum phytase was not efficacious at improving nutrient digestibility-retention in laying hens fed CSM diets deficient in nonphytate P.

Adaptive evolution of mammalian aromatases: lessons from Suiformes.

Estrogen synthesis evolved in chordates to control reproduction. The terminal enzyme in the cascade directly responsible for estrogen synthesis is aromatase cytochrome P450 (P450arom) encoded by the CYP19 gene. Mammals typically have a single CYP19 gene but pigs, peccaries and other Suiformes have two or more resulting from duplication in a common ancestor. Duplication of CYP genes in the steroid synthetic cascade has occurred for only one other enzyme, also terminal, 11beta-hydroxylase P450 (P450c11). P450arom and P450c11 share common substrates and even physiological functions as possible remnants from a common P450 progenitor, perhaps an ancestral P450arom, which is supported by phylogenetic analysis. Conserved tissue-specific expression patterns of P450arom paralogs in placenta and gonads of pigs and peccaries suggest how functional adaptation may have proceeded divergently and influenced adopted reproductive strategies including ovulation rate and litter size. Data suggest that the porcine placental paralog evolved catalytically to protect female conceptuses from testosterone produced by male siblings; the gonadal paralog to synthesize a novel, nonaromatizable testosterone metabolite (1OH-testosterone) that may increase ovulation rate. This would represent a coevolution facilitating litter bearing as pigs diverged from peccaries. Evidence of convergence between the peccary CYP19 genes and lower tissue expression may therefore represent initiation of loss of the functional paralogs. Studies on the Suiforme aromatases provide insights into the evolution of the steroidogenic cascade and metabolic pathways in general, how it translates into physiological adaptations (altered reproductive strategies for instance), and how duplicated genes become stabilized or disappear from genomes.

The efficacy of quantum phytase in a forty-week production trial using white leghorn laying hens fed corn-soybean meal-based diets.

Microbial phytase is a prominent feed enzyme used in animal feeds, but there is relatively little information on its use in laying hen diets. In this experiment, an Escherichia coli 6-phytase (Quantum) was evaluated for its efficacy in a 40-wk laying hen production trial. A total of 1,080 White Leghorn hens (540 each of Shaver and Bovan strains) were fed mash corn-soybean meal-based diets containing 0.35% (positive control, PC), 0.25% (negative control, NC1), or 0.15% (NC2) nonphytate phosphorus (NPP). Six more diets were manufactured by supplementing the negative control diets with 200, 400, and 600 U/kg of exogenous phytase, resulting in a total of 9 treatments. Each dietary treatment x strain subclass was replicated 4 times with 5 adjoining cages per replicate (3 hens per cage) in a randomized complete block design. Production performance was measured from 21 to 61 wk of age. Only minor differences in production characteristics were found between the PC and NC1 treatments regardless of phytase addition, indicating that 0.25% NPP resulted in P intake that was at or above the hen's requirement. In contrast, the hens fed 0.15% NPP diet without phytase supplementation had significantly (P < 0.05) reduced total hen housed egg production and body weight at 61 wk of age in comparison to the PC treatment, whereas the incidence of soft-shelled, cracked, and broken eggs was increased significantly (P < 0.05) in hens fed the NC2 diet. Addition of phytase to the NC2 diet improved these production characteristics to levels equal or better than the PC diet. The results indicated that Quantum phytase was efficacious in corn-soybean meal-based diets fed to White Leghorn laying hens and can be used to reduce diet supplementation with inorganic phosphorus.

Evolution of suiform aromatases: ancestral duplication with conservation of tissue-specific expression in the collared peccary (Pecari tayassu).

Aromatase cytochrome P450 (P450arom), the enzyme that catalyzes estrogen synthesis, is required for successful reproduction and is encoded by a single copy gene (CYP19) in most mammals. However, pigs and their distant suiform relatives the peccaries experienced CYP19 duplication. Here, the evolutionary origin of CYP19 duplication, and the evolution of the gene paralogs, was explored further in collared peccaries (Pecari tayassu). Exons IV and V, and the intervening intron, representing duplicated CYP19 genes, were cloned and sequenced from collared peccary, pig, and hippopotamus. Sequence alignment and analysis identified a gene conversion in collared peccary with a breakpoint 102 base pairs (bp) upstream of exon V. Phylogenetic analyses of nucleotide and amino acid sequence upstream of the breakpoint supported a tree in which one peccary sequence was orthologous with the porcine gonadal gene. Cloning and sequencing of tissue transcripts, using reverse-transcriptase polymerase chain reaction techniques (RT-PCR), confirmed that the gonadal ortholog was expressed in collared peccary testis. Orthology of the other genomic sequence with the porcine placental gene was not resolved, but its placenta-specific expression in collared peccary was confirmed by similar transcript analysis. Immunoblot and enzyme activity in collared peccary testes demonstrated much lower levels of P450arom than in pig testis. Collared peccary placental P450arom expression also seemed much lower than pigs. Thus, suiform CYP19 genes arose from an ancestral duplication that has maintained gonad- and placenta-specific expression, but at lower levels in peccaries than pigs, perhaps facilitating the emergence of different reproductive strategies as Suiformes diverged and evolved.

Looking for Darwin in all the wrong places: the misguided quest for positive selection at the nucleotide sequence level.

Recent years have seen an explosion of interest in evidence for positive Darwinian selection at the molecular level. This quest has been hampered by the use of statistical methods that fail adequately to rule out alternative hypotheses, particularly the relaxation of purifying selection and the effects of population bottlenecks, during which the effectiveness of purifying selection is reduced. A further problem has been the assumption that positive selection will generally involve repeated amino-acid changes to a single protein. This model was derived from the case of the vertebrate major histocompatibility complex (MHC), but the MHC proteins are unusual in being involved in protein-protein recognition and in a co-evolutionary process of pathogens. There is no reason to suppose that repeated amino-acid changes to a single protein are involved in selectively advantageous phenotypes in general. Rather adaptive phenotypes are much more likely to result from other causes, including single amino-acid changes; deletion or silencing of genes or changes in the pattern of gene expression.

Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta).

At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to determine the morphological and functional ancestral state of their placentas, as it relates to evolutionary relationships among species in this important taxonomic group.

Cloning of rhesus monkey LILRs.

Leukocyte Ig-like receptors (LILRs) are a family of receptors that have inhibitory and activating functions and widely expressed by lymphoid and myeloid cells. Here we report the identification of the rhesus monkey LILRs by screening of rhesus spleen and decidua cDNA libraries and RT-PCR cloning. We obtained eight different full-length clones with structural and functional diversity similar to human LILRs, including LILRs with immunoreceptor tyrosine-based inhibitory motifs, LILRs with truncated cytoplasmic tails containing positively charged arginine residues in the transmembrane domain, and putative soluble receptors lacking transmembrane or cytoplasmic domains. Characterization of rhesus LILRs will facilitate use of this non-human primate model for the study of the functional role(s) of LILRs, including immune regulation through interaction with non-classical MHC class I molecules.

Pattern and timing of gene duplication in animal genomes.

Duplication of genes, giving rise to multigene families, has been a characteristic feature of the evolution of eukaryotic genomes. In the case of vertebrates, it has been proposed that an increase in gene number resulted from two rounds of duplication of the entire genome by polyploidization (the 2R hypothesis). In the most extensive test to date of this hypothesis, we compared gene numbers in homologous families and conducted phylogenetic analyses of gene families with two to eight members in the complete genomes of Caenorhabditis elegans and Drosophila melanogaster and the available portion of the human genome. Although the human genome showed a higher proportion of recent gene duplications than the other animal genomes, the proportion of duplications after the deuterostome-protostome split was constant across families, with no peak of such duplications in four-member families, contrary to the expectation of the 2R hypothesis. A substantial majority (70.9%) of human four-member families and four-member clusters in larger families showed topologies inconsistent with two rounds of polyploidization in vertebrates.

Molecular evolution of viral fusion and matrix protein genes and phylogenetic relationships among the Paramyxoviridae.

Phylogenetic relationships among the Paramyxoviridae, a broad family of viruses whose members cause devastating diseases of wildlife, livestock, and humans, were examined with both fusion (F) and matrix (M) protein-coding sequences. Neighbor-joining trees of F and M protein sequences showed that the Paramyxoviridae was divided into the two traditionally recognized subfamilies, the Paramyxovirinae and the Pneumovirinae. Within the Paramyxovirinae, the results also showed groups corresponding to three currently recognized genera: Respirovirus, Morbillivirus, and Rubulavirus. The relationships among the three genera of the Paramyxovirinae were resolved with M protein sequences and there was significant bootstrap support (100%) showing that members of the genus Respirovirus and the genus Morbillivirus were more closely related to each other than to members of the genus Rubulavirus. Both F and M phylogenies showed that Newcastle disease virus (NDV) was more closely related to the genus Rubulavirus than to the other two genera but were consistent with the proposal (B. S. Seal et al., 2000, Virus Res. 66, 1-11) that NDV be classified as a separate genus within the Paramyxovirinae. Both F and M phylogenies were also consistent with the proposal (L. Wang et al., 2000, J. Virol 74, 9972-9979) that Hendra virus be classified as a new genus closely related and basal to the genus Morbillivirus. Rinderpest was most closely related to measles and a more derived virus than to canine distemper virus, phocine distemper virus, or dolphin morbillivirus.

Simultaneous positive and purifying selection on overlapping reading frames of the tat and vpr genes of simian immunodeficiency virus.

Tat-specific cytotoxic T cells have previously been shown to exert positive Darwinian selection favoring amino acid replacements of an epitope of simian immunodeficiency virus (SIV). The region of the tat gene encoding this epitope falls within a region of overlap between the tat and vpr reading frames, and nonsynonymous nucleotide substitutions in the tat reading frame were found to occur disproportionately in such a way as to cause synonymous changes in the vpr reading frame. Comparison of published complete SIV genomes showed Tat to be the least conserved at the amino acid level of nine proteins encoded by the virus, while Vpr was one of the most conserved. Numerous parallel amino acid changes occurred within the Tat epitope independently in different monkeys, and purifying selection on the vpr reading frame, by limiting acceptable nonsynonymous substitutions in the tat reading frame, evidently has enhanced the probability of parallel evolution.

Very large long-term effective population size in the virulent human malaria parasite Plasmodium falciparum.

It has been proposed that the virulent human malaria parasite Plasmodium falciparum underwent a recent severe population bottleneck. In order to test this hypothesis, we estimated the effective population size of this species from the patterns of nucleotide substitution at 23 nuclear protein-coding loci, using a variety of methods based on coalescent theory. Both simple methods and phylogenetically based maximum-likelihood methods yielded the conclusion that the effective population size of this species has been of the order of at least 10(5) for the past 300,000-400,000 years.

Ancient genome duplications did not structure the human Hox-bearing chromosomes.

The fact that there are four homeobox (Hox) clusters in most vertebrates but only one in invertebrates is often cited as evidence for the hypothesis that two rounds of genome duplication by polyploidization occurred early in vertebrate history. In addition, it has been observed in humans and other mammals that numerous gene families include paralogs on two or more of the four Hox-bearing chromosomes (the chromosomes bearing the Hox clusters; i.e., human chromosomes 2, 7, 12, and 17), and the existence of these paralogs has been taken as evidence that these genes were duplicated along with the Hox clusters by polyploidization. We tested this hypothesis by phylogenetic analysis of 42 gene families including members on two or more of the human Hox-bearing chromosomes. In 32 of these families there was evidence against the hypothesis that gene duplication occurred simultaneously with duplication of the Hox clusters. Phylogenies of 14 families supported the occurrence of one or more gene duplications before the origin of vertebrates, and of 15 gene duplication times estimated for gene families evolving in a clock-like manner, only six were dated to the same time period early in vertebrate history during which the Hox clusters duplicated. Furthermore, of gene families duplicated around the same time as the Hox clusters, the majority showed topologies inconsistent with their having duplicated simultaneously with the Hox clusters. The results thus indicate that ancient events of genome duplication, if they occurred at all, did not play an important role in structuring the mammalian Hox-bearing chromosomes.

Gene duplication and the structure of eukaryotic genomes.

A simple method for understanding how gene duplication has contributed to genomic structure was applied to the complete genomes of Caenorhabditis elegans, Drosophila melanogaster, and yeast Saccharomyces cerevisiae. By this method, the genes belonging to gene families (the paranome) were identified, and the extent of sharing of two or more families between genomic windows was compared with that expected under a null model. The results showed significant evidence of duplication of genomic blocks in both C. elegans and yeast. In C. elegans, the five block duplications identified all occurred intra-chromosomally, and all but one occurred quite recently. In yeast, by contrast, 39 duplicated blocks were identified, and all but one of these was inter-chromosomal. Of these 39 blocks, 28 showed evidence of ancient duplication, possibly as a result of an ancient polyploidization event. By contrast, three blocks showed evidence of very recent duplication, while seven others showed a mixture of ancient and recent duplication events. Thus, duplication of genomic blocks has been an ongoing feature of yeast evolution over the past 200--300 million years.

Evidence that intragenic recombination contributes to allelic diversity of the S-RNase gene at the self-incompatibility (S) locus in Petunia inflata.

For Solanaceae type self-incompatibility, discrimination between self and nonself pollen by the pistil is controlled by the highly polymorphic S-RNase gene. To date, the mechanism generating the allelic diversity of this gene is largely unknown. Natural populations offer a good opportunity to address this question because they likely contain different alleles that share recent common progenitors. We identified 19 S haplotypes from a natural population of Petunia inflata in Argentina, used reverse transcriptase-polymerase chain reaction to obtain cDNAs for 15 alleles of the S-RNase gene, and sequenced all the cDNAs. Phylogenetic studies revealed that five of these alleles and two previously identified alleles form a major clade, and that the 5' region of S(19) allele was derived from an ancestor allele closely related to S(2), whereas its 3' region was derived from an ancestor allele closely related to S(8). A similar evolutionary relationship was found among S(3), S(12), and S(15) alleles. These findings suggest that intragenic recombination contributed to the generation of the allelic diversity of the S-RNase gene. Two additional findings emerged from the sequence comparisons. First, the nucleotide sequence of the S(1) allele identified in this work is completely identical to that of the previously identified S(1) allele of a different origin. Second, in the two hypervariable regions HVa and HVb, thought to be involved in determining S allele specificity, S(6) and S(9) alleles differ only by four nucleotides, all in HVb, resulting in two amino acid differences. The implications of these findings are discussed.

Distinction between differentiation, cell cycle, and apoptosis signals in PC12 cells by the nerve growth factor mutant delta9/13, which is selective for the p75 neurotrophin receptor.

The common neurotrophin receptor p75(NTR) (low affinity nerve growth factor receptor) participates in the high-affinity binding with the TrkA nerve growth factor (NGF) receptor, may mediate apoptosis, and may signal independently in a cell-specific manner. The potential of p75(NTR) to signal independently of TrkA was investigated with an NGF mutant protein (NGFdelta9/13) that binds poorly to TrkA (Woo et al. [1995] J. Biol. Chem. 270:6278-6285). The NGFdelta9/13 mutant does not activate TrkA autophosphorylation and fails to stimulate the normal NGF-induced growth arrest, demonstrating that TrkA activation is required to arrest PC12 cells at the NGF-activated G1/S cell cycle checkpoint. However, apoptosis is successfully blocked and cell survival is promoted by the NGFdelta9/13 mutant in naive PC12 cells after serum withdrawal, suggesting that p75(NTR) can signal for survival autonomously of TrkA. Annexin V binding, an indication of apoptotic plasma membrane disruption, is inhibited by both NGF and the NGFdelta9/13 mutant after serum deprivation. Both NGF and the NGFdelta9/13 mutant inhibit the rapid apoptotic internucleosomal DNA cleavage of PC12 cells upon serum deprivation. Furthermore, the level of caspase3-like activity that is rapidly activated by serum withdrawal from PC12 cells is reduced by both the NGFdelta9/13 protein and NGF. Finally, upon serum withdrawal, both NGF and the NGFdelta9/13 mutant activate nuclear translocation of the transcriptional factor NF-kappaB (nuclear factor kappaB), a process involved in cell survival. These results are consistent with p75(NTR) inhibition of caspase-mediated apoptosis in PC12 cells. The different physiologic responses elicited by NGFdelta9/13 indicate the potential for individual signaling by the two NGF receptors and also demonstrate the utility of NGF mutants for receptor-selective signal transduction.