The software program TreeSAAP measures the selective influences on 31 structural and biochemical amino acid properties during cladogenesis, and performs goodness-of-fit and categorical statistical tests.

Cetaceans have most likely experienced metabolic shifts since evolutionarily diverging from their terrestrial ancestors, shifts that may be reflected in the proteins such as cytochrome b that are responsible for metabolic efficiency. However, accepted statistical methods for detecting molecular adaptation are largely biased against even moderately conservative proteins because the primary criterion involves a comparison of nonsynonymous and synonymous substitution rates (dN/dS); they do not allow for the possibility that adaptation may come in the form of very few amino acid changes. We apply the MM01 model to the possible molecular adaptation of cytochrome b among cetaceans because it does not rely on a dN/dS ratio, instead evaluating positive selection in terms of the amino acid properties that comprise protein phenotypes that selection at the molecular level may act upon. We also apply the codon-degeneracy model (CDM), which focuses on evaluating overall patterns of nucleotide substitution in terms of base exchange, codon position, and synonymy to estimate the overall effect of selection. Using these relatively new models, we characterize the molecular adaptation that has occurred in the cetacean cytochrome b protein by comparing revealed amino acid replacement patterns to those found among artiodactyls, the modern terrestrial mammals found to be most closely related to cetaceans. Our findings suggest that several regions of the cetacean cytochrome b protein have experienced molecular adaptation. Also, these adaptations are spatially associated with domain structure, protein function, and the structure and function of the cytochrome bc(1) complex and its constituents. We also have found a general correlation between the results of the analytical software programs TreeSAAP (which implements the MM01 model) and CDM (which implements the codon-degeneracy model).

A steady decline in cervical cancer incidence and mortality in the United States has been attributed to increased uptake of cervical cancer screening tests such as Papanicolau (Pap) tests. However, disparities in Pap test compliance exist, and may be due in part to perceived barriers or lack of knowledge about risk factors for cervical cancer. This study aimed to assess correlates of cervical cancer risk factor knowledge and examine socio-demographic predictors of self-reported barriers to screening among a group of low-income uninsured women. Survey and procedure data from 433 women, who received grant-funded cervical cancer screenings over a span of 33 months, were examined for this project. Data included demographics, knowledge of risk factors, and agreement on potential barriers to screening. Descriptive analysis showed significant correlation between educational attainment and knowledge of risk factors (r = 0.1381, P < 0.01). Multivariate analyses revealed that compared to Whites, Hispanics had increased odds of identifying fear of finding cancer (OR 1.56, 95% CI 1.00–2.43), language barriers (OR 4.72, 95% CI 2.62–8.50), and male physicians (OR 2.16, 95% CI 1.32–3.55) as barriers. Hispanics (OR 1.99, 95% CI 1.16–3.44) and Blacks (OR 2.06, 95% CI 1.15–3.68) had a two-fold increase in odds of agreeing that lack of knowledge was a barrier. Identified barriers varied with age, marital status and previous screening. Programs aimed at conducting free or subsidized screenings for medically underserved women should include culturally relevant education and patient care in order to reduce barriers and improve screening compliance for safety-net populations.

The unwitting inclusion of convergent characters in phylogenetic estimates poses a serious problem for efforts to recover phylogeny. Convergence is not inscrutable, however, particularly when one group of characters tracks phylogeny and another set tracks adaptive history. In such cases, convergent characters may be correlated with one or a few functional anatomical units and readily identifiable by using comparative methods. Stifftail ducks (Oxyurinae) offer one such opportunity to study correlated character evolution and function in the context of phylogenetic reconstruction. Morphological analyses place stifftail ducks as part of a large clade of diving ducks that includes the sea ducks (Mergini), Hymenolaimus, Merganetta, and Tachyeres, and possibly the pochards (Aythyini). Molecular analyses, on the other hand, place stifftails far from other diving ducks and suggest, moreover, that stifftails are polyphyletic. Mitochondrial cytochrome b gene sequences of eight stifftail species traditionally supposed to form a clade were compared with each other and with sequences from 50 other anseriform and galliform species. Stifftail ducks are not the sister group of sea ducks but lie outside the typical ducks (Anatinae). Of the four traditional stifftail genera, monophyly of Oxyura and its sister group relationship with Nomonyx are strongly supported. Heteronetta probably is the sister group of that clade, but support is weak. Biziura is not a true stifftail. Within Oxyura, Old World species (O. australis, O. leucocephala, O. maccoa) appear to form a clade, with New World species (O. jamaicensis, O. vittata) branching basally. Incongruence between molecules and morphology is interpreted to be the result of adaptive specialization and functional convergence in the hind limbs of Biziura and true stifftails. When morphological characters are divided into classes, only hind-limb characters are significantly in conflict with the molecular tree. Likewise, null models of synonymous and nonsynonymous substitution based on patterns of codon-degeneracy and chemical dissimilarity indicate that the nucleotide and amino acid changes postulated by the molecular tree are more plausible than those postulated by the morphological tree. These findings teach general lessons about the utility of highly adaptive characters (in particular those related to foraging ecology) and underscore the problems that convergence can pose for attempts to recover phylogeny. They also demonstrate how the concept of natural data partitions and simple models of evolution (e.g., parsimony, likelihood, neutrality) can be used to test the accuracy of independent phylogenetic estimates and provide arguments in favor of one tree topology over another.

We evaluated the effects of selection on the molecular evolution of the functional domains of the mammalian cytochrome b gene as it relates to physicochemical properties shown to correlate with rates of amino acid replacement. Two groups of mammals were considered: pocket gophers of the rodent family Geomyidae, and cetaceans and ungulates of the monophyletic taxon Cetartopdactyla. Several characteristics of cytochrome b evolution were common to both mammal groups. The evolution of the matrix domain reflected the region's relative lack of function. Goodness of fit to neutral expectations indicated that external influences have had very little effect on the evolution of the matrix, although in some cases conservative and moderate changes have been favored. Although rates of synonymous nucleotide substitution have been relatively high, the transmembrane domain exhibited poor goodness of fit to neutral expectations. However, the evolution of the transmembrane domain has been constrained by negative selection, allowing a preponderance of conservative and moderate amino acid replacements. We hypothesize that a high rate of substitution is maintained in spite of negative selection because the codons of the transmembrane coding region are predisposed to conservative changes in all amino acid properties. The evolutionary patterns of the intermembrane domain in pocket gophers and cetartiodactyls, however, were very different. Changes inferred from the pocket gopher phylogenetic tree exhibited a significant fit to neutral expectations for each of the amino acid properties. Changes inferred from the cetartiodactyl tree exhibited significant fit to neutral expectations for polarity and isoelectric point, but not for composition, molecular volume, polar requirement, or hydropathy. In each case, lack of fit was due to selection that promoted conservative or moderate change, with the noteworthy exception of polar requirement. We detected an unexpectedly large change in polar requirement (from aspartic acid to threonine) in two separate lineages (Camelus bactrianus and all cetaceans) at amino acid position 159. This inferred change occurred in a region of the cyt-b protein that directly interacts with external surface proteins of the cytochrome bc(1) complex and resulted in a reversion to a more common character state in vertebrates.

Investigations of opsin evolution outside of vertebrate systems have long been focused on insect visual pigments, whereas other groups have received little attention. Furthermore, few studies have explicitly investigated the selective influences across all the currently characterized arthropod opsins. In this study, we contribute to the knowledge of crustacean opsins by sequencing 1 opsin gene each from 6 previously uncharacterized crustacean species (Euphausia superba, Homarus gammarus, Archaeomysis grebnitzkii, Holmesimysis costata, Mysis diluviana, and Neomysis americana). Visual pigment spectral absorbances were measured using microspectrophotometry for species not previously characterized (A. grebnitzkii=496 nm, H. costata=512 nm, M. diluviana=501 nm, and N. americana=520 nm). These novel crustacean opsin sequences were included in a phylogenetic analysis with previously characterized arthropod opsin sequences to determine the evolutionary placement relative to the well-established insect spectral clades (long-/middle-/short-wavelength sensitive). Phylogenetic analyses indicate these novel crustacean opsins form a monophyletic clade with previously characterized crayfish opsin sequences and form a sister group to insect middle-/long-wavelength-sensitive opsins. The reconstructed opsin phylogeny and the corresponding spectral data for each sequence were used to investigate selective influences within arthropod, and mainly "pancrustacean," opsin evolution using standard dN/dS ratio methods and more sensitive techniques investigating the amino acid property changes resulting from nonsynonymous replacements in a historical (i.e., phylogenetic) context. Although the conservative dN/dS methods did not detect any selection, 4 amino acid properties (coil tendencies, compressibility, power to be at the middle of an alpha-helix, and refractive index) were found to be influenced by destabilizing positive selection. Ten amino acid sites relating to these properties were found to face the binding pocket, within 4 A of the chromophore and thus have the potential to affect spectral tuning.

We use a multigene data set (the mitochondrial locus and nine nuclear gene regions) to test phylogenetic relationships in the South American "lava lizards" (genus Microlophus) and describe a strategy for aligning noncoding sequences that accounts for differences in tempo and class of mutational events. We focus on seven nuclear introns that vary in size and frequency of multibase length mutations (i.e., indels) and present a manual alignment strategy that incorporates insertions and deletions (indels) for each intron. Our method is based on mechanistic explanations of intron evolution that does not require a guide tree. We also use a progressive alignment algorithm (Probabilistic Alignment Kit; PRANK) and distinguishes insertions from deletions and avoids the "gapcost" conundrum. We describe an approach to selecting a guide tree purged of ambiguously aligned regions and use this to refine PRANK performance. We show that although manual alignment is successful in finding repeat motifs and the most obvious indels, some regions can only be subjectively aligned, and there are limits to the size and complexity of a data matrix for which this approach can be taken. PRANK alignments identified more parsimony-informative indels while simultaneously increasing nucleotide identity in conserved sequence blocks flanking the indel regions. When comparing manual and PRANK with two widely used methods (CLUSTAL, MUSCLE) for the alignment of the most length-variable intron, only PRANK recovered a tree congruent at deeper nodes with the combined data tree inferred from all nuclear gene regions. We take this concordance as an objective function of alignment quality and present a strongly supported phylogenetic hypothesis for Microlophus relationships. From this hypothesis we show that (1) a coded indel data partition derived from the PRANK alignment contributed significantly to nodal support and (2) the indel data set permitted detection of significant conflict between mitochondrial and nuclear data partitions, which we hypothesize arose from secondary contact of distantly related taxa, followed by hybridization and mtDNA introgression.

Abstract-The TreeSAAP software has been successfully used in a variety of protein studies for identifying and characterizing adaptation in terms of shifts in the physicochemical properties of amino acid replacements. It differentiates adaptive replacements from those that may have resulted from random mutation. The accuracy of TreeSAAP was tested using simulated protein-coding DNA data that was randomly generated using a bifurcating phylogeny to reflect a random pattern of mutation constrained only by the structure of the genetic code. A sampling of 1402 simulated amino acid replacements resulted in a default accuracy of 80.6%. More than 50% of the false-positive results were traced to just 11 of the possible single-step amino acid exchanges, each of which exhibited less than 50% accuracy. When these 11 exchanges are eliminated from the subsequent analysis, the accuracy of TreeSAAP is increased to nearly 90%. Further testing of this modified approach for adverse implications with empirical data is warranted.