Heterogeneity and concordance in locus-specific differentiation and introgression between species of towhees.

The maintenance or breakdown of reproductive isolation is an observable outcome of secondary contact between species. In cases where hybrids beyond the F1 are formed, the representation of each species' ancestry can vary dramatically among genomic regions. This genomic heterogeneity in ancestry and introgression can offer insight into evolutionary processes, particularly if introgression is compared in multiple hybrid zones. Similarly, considerable heterogeneity exists across the genome in the extent to which populations and species have diverged, reflecting the combined effects of different evolutionary processes on genetic variation. We studied hybridization across two hybrid zones of two phenotypically well-differentiated bird species in Mexico (Pipilo maculatus and P. ocai), to investigate genomic heterogeneity in differentiation and introgression. Using genotyping-by-sequencing (GBS) and hierarchical Bayesian models, we genotyped 460 birds at over 41 000 single nucleotide polymorphism (SNP) loci. We identified loci exhibiting extreme introgression relative to the genome-wide expectation using a Bayesian genomic cline model. We also estimated locus-specific FST and identified loci with exceptionally high genetic divergence between the parental species. We found some concordance of locus-specific introgression in the two independent hybrid zones (6-20% of extreme loci shared across zones), reflecting areas of the genome that experience similar gene flow when the species interact. Additionally, heterogeneity in introgression and divergence across the genome revealed another subset of loci under the influence of locally specific factors. These results are consistent with a history in which reproductive isolation has been influenced by a common set of loci in both hybrid zones, but where local environmental and stochastic factors also lead to genomic differentiation.

The genomic consequences of adaptive divergence and reproductive isolation between species of manakins.

The processes of adaptation and speciation are expected to shape genomic variation within and between diverging species. Here we analyze genomic heterogeneity of genetic differentiation and introgression in a hybrid zone between two bird species (Manacus candei and M. vitellinus) using 59 100 SNPs, a whole genome assembly, and Bayesian models. Measures of genetic differentiation (FST) and introgression (genomic cline center [α] and rate [ß]) were highly heterogeneous among loci. We identified thousands of loci with elevated parameter estimates, some of which are likely to be associated with variation in fitness in Manacus populations. To analyze the genomic organization of differentiation and introgression, we mapped SNPs onto a draft assembly of the M. vitellinus genome. Estimates of FST, α, and ß were autocorrelated at very short physical distances (< 100 bp), but much less so beyond this. In addition, average statistical associations (linkage disequilibrium) between SNPs were generally low and were not higher in admixed populations than in populations of the parental species. Although they did not occur with a constant probability across the genome, loci with elevated FST, α, and ß were not strongly co-localized in the genome. Contrary to verbal models that predict clustering of loci involved in adaptation and isolation in discrete genomic regions, these results are consistent with the hypothesis that genetic regions involved in adaptive divergence and reproductive isolation are scattered throughout the genome. We also found that many loci were characterized by both exceptional genetic differentiation and introgression, consistent with the hypothesis that loci involved in isolation are also often characterized by a history of divergent selection. However, the concordance between isolation and differentiation was only partial, indicating a complex architecture and history of loci involved in isolation.

Population genetic structure and its implications for adaptive variation in memory and the hippocampus on a continental scale in food-caching black-capped chickadees.

Food-caching birds rely on stored food to survive the winter, and spatial memory has been shown to be critical in successful cache recovery. Both spatial memory and the hippocampus, an area of the brain involved in spatial memory, exhibit significant geographic variation linked to climate-based environmental harshness and the potential reliance on food caches for survival. Such geographic variation has been suggested to have a heritable basis associated with differential selection. Here, we ask whether population genetic differentiation and potential isolation among multiple populations of food-caching black-capped chickadees is associated with differences in memory and hippocampal morphology by exploring population genetic structure within and among groups of populations that are divergent to different degrees in hippocampal morphology. Using mitochondrial DNA and 583 AFLP loci, we found that population divergence in hippocampal morphology is not significantly associated with neutral genetic divergence or geographic distance, but instead is significantly associated with differences in winter climate. These results are consistent with variation in a history of natural selection on memory and hippocampal morphology that creates and maintains differences in these traits regardless of population genetic structure and likely associated gene flow.

Unidirectional spread of secondary sexual plumage traits across an avian hybrid zone.

Theory predicts that traits under positive selection can rapidly cross a hybrid zone in spite of a substantial barrier to neutral gene flow between hybridizing taxa. An avian hybrid zone between Manacus candei (white-collared manakin) and M. vitellinus (golden-collared manakin) is reported here that displays an unusual pattern of noncoincident clines. Male secondary sexual traits of M. vitellinus have spread into populations that are genetically and morphometrically like M. candei. These birds have a lek breeding system in which male mating success is highly skewed, suggesting that sexual selection is driving male sexual traits across the zone.

Sexual selection on plumage and behavior in an avian hybrid zone: experimental tests of male-male interactions.

In western Panama, an unusual hybrid zone exists between white-collared manakins, Manacus candei, and golden-collared manakins, M. vitellinus. Unidirectional introgression of plumage traits from vitellinus into candei has created a region in which all definitively plumaged males have a collar that is lemon-colored. These males are nearly indistinguishable from white-collared candei genetically and morphometrically, but strongly resemble golden-collared vitellinus due to the introgression of secondary sexual plumage traits, particularly the lemon-colored collar. The introgression could be explained by sexual selection for golden-collared traits or by a series of mechanisms that do not invoke sexual selection (e.g., neutral diffusion, dominant allele). Sexual selection on male-male interactions implies behavioral differences among the plumage forms--specifically that golden- and lemon-collared males should be more aggressive than white-collared males. In contrast, the nonsexual hypotheses predict behavioral similarity between lemon- and white-collared males, based on their nearly identical genetics. We tested the sexual selection hypothesis experimentally, by presenting males with taxidermic mounts of the three forms. As response variables, we monitored vocalizations and attacks on the mounts by replicate subject males. Both golden-collared and lemon-collared males were more likely to attack than were white-collared males, as predicted under sexual selection but not by the nonsexual hypotheses. Lemon-collared males were more vocally reactive than either parental form, contrary to the prediction of the nonsexual hypotheses. Our study demonstrates that sexual selection on male-male interactions may play an important role in the dynamics of character evolution and hybrid zones.

Evolutionary implications of divergent clines in an avian (Manacus: Aves) hybrid zone.

A previous study of the hybrid zone in western Panama between white-collared (Manacus candei) and golden-collared manakins (M. vitellinus) documented the unidirectional introgression of vitellinus male secondary sexual traits across the zone. Here, we examine the hybrid zone in greater genetic and morphological detail. Statistical comparisons of clines are performed using maximum-likelihood and nonparametric bootstrap methods. Our results demonstrate that an array of six molecular and two morphometric markers agree in cline position and width. Clines for male collar and belly color are similar in width to the first eight clines, but are shifted in position by at least five cline widths. The result is that birds in intervening populations are genetically and morphometrically very like parental candei, but males have the plumage color of parental vitellinus. Neither neutral diffusion nor nonlinearity of color scales appear to be viable explanations for the large cline shifts. Genetic dominance of vitellinus plumage traits is another potential explanation that will require breeding experiments to test. Sexual selection remains a plausible explanation for the observed introgression of vitellinus color traits in these highly dimorphic, polygynous, lek-mating birds. Two other clines, including a nondiagnostic isozyme locus, are similar in position to the main cluster of clines, but are broader in width. Thus, introgression at some loci is greater than that detected with diagnostic markers. Assuming that narrow clines are maintained by selection, variation in cline width indicates that selection is not uniform throughout the genome and that diagnostic markers are under more intense selective pressure. The traditional focus on diagnostic markers in studies of hybrid zones may therefore lead to underestimates of average introgression. This effect may be more pronounced in organisms with low levels of genetic divergence between hybridizing taxa.

Increasing proportions of uracil in DNA substrates increases inhibition of restriction enzyme digests.

Techniques that rely upon the incorporation of uracil into DNA are being published with increasing frequency, especially in PCR protocols. We report here the efficiency of 18 type II restriction enzymes to digest PCR amplicons synthesized with varying proportions of TTP to dUTP in the PCR mixture. We find that most enzymes with A:T/U bp in their recognition site digest the amplicons less efficiently as the percentage of dUTP in the reaction mixture is increased. This effect is most dramatic when the proportion of dUTP in the nucleotide mixture exceeds 50%. All but one of the enzymes which fail to digest amplicons that are synthesized with 100% dUTP digest some amplicons which are synthesized with 90% dUTP.

Phylogenetic relationships of the thylacine (Mammalia: Thylacinidae) among dasyuroid marsupials: evidence from cytochrome b DNA sequences.

DNA sequences from the mitochondrial cytochrome b gene were obtained from a museum specimen of the presumed extinct thylacine (Thylacinus cynocephalus) and were compared with homologous sequences from 13 representatives of the Australian marsupial family Dasyuridae. The relationship of the thylacine to dasyurids has been suggested by previous anatomical and molecular studies, but its position within the dasyuroid radiation has not been addressed with genetic data. Phylogenetic analysis of the sequences reported here suggests that the thylacine is a sister group to Dasyuridae and lends support to the hypothesis that Thylacinus represents an ancient Australian marsupial lineage. Relationships with Dasyuridae support the results of other recent molecular studies, particularly in showing the affinities of endemic New Guinean subfamilies with larger Australian clades.

Clinical evaluation of a high-resolution new peripheral quantitative computerized tomography (pQCT) scanner for the bone densitometry at the lower limbs.

Precision, long-term stability, linearity and accuracy of the x-ray peripheral quantitative computerized tomographic (pQCT) bone scanner XCT 3000 (Norland-Stratec Medical Sys.) were evaluated using the European Forearm Phantom (EFP). In vivo measurements were assessed using a standardized procedure at the distal femur and the distal tibia. In the patient-scan mode, the spatial resolution of the system was 1.04 +/- 0.05 lp/mm as measured at the 10% level of the modulation transfer function (MTF). The contrast-detail diagram (CDD) yielded a minimal difference in attenuation coefficient (AC) of 0.07 cm(-1) at an object size of 0.5 mm. The effective dose for humans was calculated to be less than 1.5 microSv per scan. Short-term precision in vivo was expressed as root mean square standard deviation of paired measurements of 20 healthy volunteers (RMSSD = 0.5%). At the distal femur total volumetric density (ToD) and total cross-sectional area (ToA) were found to be less sensitive to positioning errors than at the distal tibia. Structural parameters like the polar cross-sectional moment of inertia (CSMIp) or the polar cross-sectional moment of resistance (CSMRp) showed a good short-term precision at the distal femur (RMSSD = 1.2 and 1.4%). The relation between the two skeletal sites with respect to CSMIp or CSMRp showed a high coefficient of determination (r2 = 0.77 and 0.74).

A molecular phylogenetic survey of the nightjars and allies (Caprimulgiformes) with special emphasis on the potoos (Nyctibiidae).

A 656-pb fragment of the mitochondrial cytochrome b gene was sequenced for six species of Central and South American potoos (genus Nyctibius, Nyctibiidae) as well as for selected representatives of all other caprimulgiform families. Sequence divergence among potoos was much higher (11.1-16.2%) than has typically been observed among congeneric species of birds, suggesting that the species of Nyctibius are quite old. Divergence among families was also quite high (13.7-21.8%), confirming recent DNA-DNA hybridization studies. Such high genetic divergences in a functionally constrained gene like cyt b indicate that many of those sites which are free to vary will have experienced multiple substitutions. We therefore performed phylogenetic analysis using parsimony under a variety of weighting schemes intended to reduce the effect of multiple substitutions. The monophyly of all the traditional caprimulgiform families was confirmed and a number of new hypotheses of relationship emerged. From our analysis, it appears that the oilbird (Steatornis) is an ancient member of the order, and it is not closely related to the potoos. We also note the close link between Aegothelidae and Caprimulgidae, and the basal position of the Podargidae/Batrachostomidae clade in the phylogeny. These results are in accordance with several classical works of the first half of the century. The relationships of the various Nyctibius species to each other have not been fully resolved; however, a close link between N. leucopterus and N. maculosus appears to be highly probable from our data.

The visna virus genome: evidence for a hypervariable site in the env gene and sequence homology among lentivirus envelope proteins.

The complete nucleotide sequence of the visna virus 1514 genome was determined. Our sequence confirms the relationship of visna virus and other lentiviruses to human immunodeficiency virus (HIV) both at the level of sequence homology and of genomic organization. Sequence homology is shown to extend to the transmembrane proteins of lentivirus env genes; this homology is strongest in the extracellular domain, suggesting that close structural and functional similarities may also exist among these envelope proteins. Comparison of our data with the sequence of visna virus LV1-1, an antigenic variant derived from strain 1514, demonstrates that the rate of divergence has been about 1.7 x 10(-3) substitutions per nucleotide per year in vivo. This rate is orders of magnitude higher than that for most DNA genomes, but agrees well with estimates of the rate for HIV. A statistically significant cluster of mutations in the env gene appears to represent a hypervariable site and may correspond to the epitope responsible for the antigenic differences between 1514 and LV1-1. Analysis of the potential RNA folding pattern of the visna virus env gene shows that this hypervariable site falls within a region with little potential for intramolecular base pairing. This correlation of hypervariability with lack of RNA secondary structure is strengthened by the fact that it also holds for a hypervariable site in the env gene of HIV.

Nucleotide sequence of a transduced myc gene from a defective feline leukemia provirus.

The nucleotide sequence of a feline v-myc gene and feline leukemia virus (FeLV) flanking regions was determined. Both the nucleotide and predicted amino acid sequences are very similar to the murine and human c-myc genes (ca. 90% identity). The entire c-myc coding sequence is represented in feline v-myc and replaces portions of the gag and env genes and the entire pol gene. The coding sequence is in phase with the gag gene reading frame; v-myc, therefore, appears to be expressed as a gag-myc fusion protein. Viral sequences at the 3' myc-FeLV junction begin with the hexanucleotide CTCCTC, which is also found at the 3' fes-FeLV junction of both Gardner-Arnstein and Snyder-Theilen feline sarcoma viruses. These similarities suggest that some sequence specificity may exist for the transduction of cellular genes by FeLV. Feline v-myc lacks a potential phosphorylation site at amino acid 343 in the putative DNA-binding domain, whereas both human and murine c-myc have such sites. Avian v-myc has lost a potential phosphorylation site which is present in avian c-myc five amino acids from the potential mammalian site. If these sites are actually phosphorylated in normal c-myc proteins, their loss may alter the DNA-binding affinity of v-myc proteins.

Random amplified polymorphic DNA (RAPD) markers readily distinguish cryptic mosquito species (Diptera: Culicidae: Anopheles).

The usefulness of random amplified polymorphic DNA (RAPD) was examined as a potential tool to differentiate cryptic mosquito species. It proved to be a quick, effective means of finding genetic markers to separate two laboratory populations of morphologically indistinguishable African malaria vectors, Anopheles gambiae and An. arabiensis. In an initial screening of fifty-seven RAPD primers, 377 bands were produced, 295 of which differed between the two species. Based on criteria of interpretability, simplicity and reproducibility, thirteen primers were chosen for further screening using DNA from thirty individuals of each species. Seven primers produced diagnostic bands, five of which are described here. Some problematic characteristics of RAPD banding patterns are discussed and approaches to overcome these are suggested.

Stability of RNA stem-loop structure and distribution of non-random structure in the human immunodeficiency virus (HIV-I).

The stability of potential RNA stem-loop structures in human immunodeficiency virus isolates, HTLV-III and ARV, has been calculated, and the relevance to the local significant secondary structures in the sequence has been tested statistically using a Monte Carlo simulation method. Potentially significant structures exist in the 5'non-coding region, the boundary regions between the protein coding frames, and the 3' non-coding region. The locally optimal secondary structure occurring in the 5' terminal region has been assessed using different overlapping segment sizes and the Monte Carlo method. The results show that the most favorable structure for the 5' mRNA leader sequence of HIV has two stem-loops folded at nucleotides 5-104 in the R region (stem-loop I, 5-54 and stem-loop II, 58-104). A large fluctuation of segment score of the local optimal secondary structure also occurs in the boundary between the exterior glycosylated protein or outer membrane protein and transmembrane protein coding region. This finding is surprising since no RNA signals or RNA processing are expected to occur at this site. In addition, regions of the genome predicted to have significantly more open structure at the RNA level correlate closely with hypervariable sites found in these viral genomes. The possible importance of local secondary structure to the biological function of the human immunodeficiency virus genome is discussed.