Application and accuracy of molecular phylogenies.

Molecular investigations of evolutionary history are being used to study subjects as diverse as the epidemiology of acquired immune deficiency syndrome and the origin of life. These studies depend on accurate estimates of phylogeny. The performance of methods of phylogenetic analysis can be assessed by numerical simulation studies and by the experimental evolution of organisms in controlled laboratory situations. Both kinds of assessment indicate that existing methods are effective at estimating phylogenies over a wide range of evolutionary conditions, especially if information about substitution bias is used to provide differential weightings for character transformations.

Phylogeography and historical ecology of the North Atlantic intertidal.

Recent glaciation covered the full extent of rocky intertidal habitat along the coasts of New England and the Canadian Maritimes. To test whether this glaciation in fact caused wholesale extinction of obligate rocky intertidal invertebrates, and thus required a recolonization from Europe, we compared American and European populations using allelic diversity and techniques adapted from coalescent theory. Mitochondrial DNA sequences were collected from amphi-Atlantic populations of three cold-temperate obligate rocky intertidal species (a barnacle, Semibalanus balanoides, and two gastropods, Nucella lapillus and Littorina obtusata) and three cold-temperate habitat generalist species (a seastar, Asterias rubens; a mussel, Mytilus edulis, and an isopod, Idotea balthica). For many of these species we were able to estimate the lineage-specific mutation rate based on trans-Arctic divergences between Pacific and Atlantic taxa. These data indicate that some obligate rocky intertidal taxa have colonized New England from European populations. However, the patterns of persistence in North America indicate that other life-history traits, including mech anisms of dispersal, may be more important for surviving dramatic environmental and climatic change.

Independent contrasts succeed where ancestor reconstruction fails in a known bacteriophage phylogeny.

Methods of ancestor reconstruction are important tools for evolutionary inference that are difficult to test empirically because ancestral states are rarely known with certainty. We evaluated reconstruction methods for continuous phenotypic characters using taxa from an experimentally generated bacteriophage phylogeny. Except for one slowly evolving character, the estimated ancestral states of continuous phenotypic characters were highly inaccurate and biased, even when including a known ancestor at the root. This error was caused by a directional trend in character evolution and by rapid rates of character evolution. Computer simulations confirmed that such factors affect reconstruction of continuous characters in general. We also used phenotypic viral characters to evaluate two methods that attempt to estimate the correlation between characters during evolution. Whereas a nonphylogenetic regression was relatively inaccurate and biased, independent contrasts accurately estimated the correlation between characters with little bias.

A comparative study of asymmetric migration events across a marine biogeographic boundary.

In many nonclonal, benthic marine species, geographic distribution is mediated by the dispersal of their larvae. The dispersal and recruitment of marine larvae may be limited by temperature gradients that can affect mortality or by ocean currents that can directly affect the movements of pelagic larvae. We focus on Point Conception, a well-known biogeographic boundary between the Californian and Oregonian biogeographic provinces, to investigate whether ocean currents affect patterns of gene flow in intertidal marine invertebrates. The predominance of pelagically dispersing species with northern range limits at Point Conception suggests that ocean currents can affect species distributions by erecting barriers to the dispersal of planktonic larvae. In this paper, we investigate whether the predominantly southward currents have left a recognizable genetic signature in species with pelagically dispersing larvae whose ranges span Point Conception. We use patterns of genetic diversity and a new method for inferring cladistic migration events to test the hypothesis that southward currents increase southward gene flow for species with pelagically dispersing larvae. We collected mitochondrial DNA (mtDNA) sequence data for the barnacles Balanus glandula and Chthamalus fissus and also reanalyzed a previously published mtDNA dataset (Strongylocentrotus purpuratus, Edmands et al. 1996). For all three species, our cladistic approach identified an excess of southward migration events across Point Conception. In data from a fourth species with nondispersing larvae (Nucella emarginata, Marko 1998), our method suggests that ocean currents have not played a role in generating genetic structure.

Mitochondrial gene rearrangements confirm the parallel evolution of the crab-like form.

The repeated appearance of strikingly similar crab-like forms in independent decapod crustacean lineages represents a remarkable case of parallel evolution. Uncertainty surrounding the phylogenetic relationships among crab-like lineages has hampered evolutionary studies. As is often the case, aligned DNA sequences by themselves were unable to fully resolve these relationships. Four nested mitochondrial gene rearrangements--including one of the few reported movements of an arthropod protein-coding gene--are congruent with the DNA phylogeny and help to resolve a crucial node. A phylogenetic analysis of DNA sequences, and gene rearrangements, supported five independent origins of the crab-like form, and suggests that the evolution of the crab-like form may be irreversible. This result supports the utility of mitochondrial gene rearrangements in phylogenetic reconstruction.

Risk factors for respiratory syncytial virus bronchiolitis hospital admission in New Zealand.

This study assessed risk factors for respiratory syncytial virus (RSV) hospitalization and disease severity in Wellington, New Zealand. During the southern hemisphere winter months of 2003--2005, 230 infants aged < 24 months hospitalized with bronchiolitis were recruited. RSV was indentified in 141 (61%) infants. Comparison with data from all live hospital births from the same region (2003--2005) revealed three independent risk factors for RSV hospitalization: birth between February and July [adjusted risk ratio (aRR) 1.62, 95% confidence interval (CI) 1.5-2.29], gestation <37 weeks (aRR 2.29, 95% CI 1.48-3.56) and Maori ethnicity (aRR 3.64, 95% CI 2.27-5.85), or Pacific ethnicity (aRR 3.60, 95% CI 2.14-6.06). The high risk for Maori and Pacific infants was only partially accounted for by other known risk factors. This work highlights the importance of RSV disease in indigenous and minority populations, and identifies the need for further research to develop public health measures that can reduce health disparities.

Association between glycosylated hemoglobin and cancer risk: a New Zealand linkage study.

BACKGROUND: The purpose of this study was to examine the relationship between glycosylated hemoglobin (HbA(1c)) level and subsequent cancer risk. MATERIAL AND METHODS: HbA(1c) measurements were made on blood samples of participants in a hepatitis B (HB) screening program (1999-2001). Cancer incidence was determined by linkage to cancer registrations and hospitalization records to the end of 2004. Participants previously diagnosed with diabetes or cancer were excluded. Hazard ratios (HR) and 95% confidence intervals (CIs) were estimated using Cox regression. RESULTS: Among the 46 575 participants (70% Maori, 12% Pacific, 5% Asian and 12% Other), 634 cancer cases were observed. For all cancers combined, a significant increased risk was found in persons with moderately elevated HbA(1c) levels (6%-6.9%) (HR 1.40, 95% CI: 1.11-1.76), with a smaller increased risk in persons with highly elevated levels (> or =7%) (HR 1.09, 95% CI: 0.80-1.48) as compared with persons having low HbA(1c) levels (<6%). The HRs for respiratory cancers were 2.27 (95% CI: 1.34-3.86) for the moderate HbA(1c) category and 1.58 (95% CI: 0.77-3.26) for the upper HbA(1c) category. For endometrial cancers, the HRs were 4.05 (95% CI: 1.10-14.88) and 5.07 (95% CI: 1.20-21.31), respectively. For other cancer sites, no significantly increased risks were found. CONCLUSIONS: These findings are consistent with other evidence that abnormal glucose metabolism may be associated with an increased risk of some cancers.

Insulin sensitivity and intramyocellular lipid concentrations in young Maori men.

AIMS: In muscle, resistance to insulin-mediated glucose uptake is thought to underlie the pre-Type 2 diabetic condition. In European Caucasian men, insulin sensitivity is negatively associated with intramyocellular lipid (IMCL) content, and this may provide an early marker of diabetes risk. This study was designed to examine the relationship between vastus lateralis IMCL content, aerobic fitness, adiposity and markers of insulin sensitivity in healthy, young Maori men. METHODS: The following parameters were measured in 24 Maori men aged 28 +/- 6 years (mean +/- sd): overnight fasting blood concentrations of glucose, insulin and triglycerides; body composition using underwater weighing; maximal oxygen uptake (VO2max) using an incremental treadmill exercise test; and vastus lateralis IMCL concentration using proton nuclear magnetic resonance spectroscopy (1H MRS). RESULTS: All participants had normal fasting blood glucose. Simultaneous multiple regression analysis with homeostasis model assessment-insulin resistance (HOMA-IR) as the dependent variable showed that: (i) increased body fatness and decreased aerobic capacity (per kg lean body mass) are significant predictors of insulin sensitivity as estimated by HOMA-IR; and (ii) although vastus lateralis IMCL concentrations are elevated, they do not contribute to the prediction of insulin sensitivity. CONCLUSIONS: IMCL is not a reliable marker of estimated insulin resistance in this cohort of young, healthy Maori men. However, measures of composition and aerobic fitness may be of use as non-invasive, culturally acceptable measures to help identify Maori men with impaired insulin action, but normal fasting glycaemia.

Nearshore fish (Pholis gunnellus) persists across the North Atlantic through multiple glacial episodes.

The intertidal biota of the North Atlantic is characterized by two disjunct communities (North American and European) exposed to different climatic regimes during the Pleistocene and in the Holocene. We collect multilocus DNA sequence data from the nearshore fish Pholis gunnellus to help uncover processes determining biogeographical persistence during periodic coastal glaciations. Coalescent-based estimates from the multilocus DNA sequence data suggest that P. gunnellus persisted on both sides of the North Atlantic throughout the last two glacial maxima (> 202,000 years) with little trans-Atlantic gene flow since divergence, very little structure among populations within Europe (Phi(ST) < 0.05) and some structure within the North American coastline (Phi(ST) = 0.0-0.21). Although the ecological flexibility and high local migration of P. gunnellus could have enhanced this species' survival across the Atlantic, logistic regression did not find a significant determinant of trans-Atlantic persistence when considering 12 other North Atlantic phylogeographical studies from the literature.

Local adaptation and species segregation in two mussel (Mytilus edulis x Mytilus trossulus) hybrid zones.

Few marine hybrid zones have been studied extensively, the major exception being the hybrid zone between the mussels Mytilus edulis and Mytilus galloprovincialis in southwestern Europe. Here, we focus on two less studied hybrid zones that also involve Mytilus spp.; Mytilus edulis and Mytilus trossulus are sympatric and hybridize on both western and eastern coasts of the Atlantic Ocean. We review the dynamics of hybridization in these two hybrid zones and evaluate the role of local adaptation for maintaining species boundaries. In Scandinavia, hybridization and gene introgression is so extensive that no individuals with pure M. trossulus genotypes have been found. However, M. trossulus alleles are maintained at high frequencies in the extremely low salinity Baltic Sea for some allozyme genes. A synthesis of reciprocal transplantation experiments between different salinity regimes shows that unlinked Gpi and Pgm alleles change frequency following transplantation, such that post-transplantation allelic composition resembles native populations found in the same salinity. These experiments provide strong evidence for salinity adaptation at Gpi and Pgm (or genes linked to them). In the Canadian Maritimes, pure M. edulis and M. trossulus individuals are abundant, and limited data suggest that M. edulis predominates in low salinity and sheltered conditions, whereas M. trossulus are more abundant on the wave-exposed open coasts. We suggest that these conflicting patterns of species segregation are, in part, caused by local adaptation of Scandinavian M. trossulus to the extremely low salinity Baltic Sea environment.

Can three incongruence tests predict when data should be combined?

Advocates of conditional combination have argued that testing for incongruence between data partitions is an important step in data exploration. Unless the partitions have had distinct histories, as in horizontal gene transfer, incongruence means that one or more data support the wrong phylogeny. This study examines the relationship between incongruence and phylogenetic accuracy using three tests of incongruence. These tests were applied to pairs of mitochondrial DNA data partitions from two well-corroborated vertebrate phylogenies. Of the three tests, the most useful was the incongruence length difference test (ILD, also called the partition homogeneity test). This test distinguished between cases in which combining the data generally improved phylogenetic accuracy (P > 0.01) and cases in which accuracy of the combined data suffered relative to the individual partitions (P < 0.001). In contrast, in several cases, the Templeton and Rodrigo tests detected highly significant incongruence (P < 0.001) even though combining the incongruent partitions actually increased phylogenetic accuracy. All three tests identified cases in which improving the reconstruction model would improve the phylogenetic accuracy of the individual partitions.

Is congruence between data partitions a reliable predictor of phylogenetic accuracy? Empirically testing an iterative procedure for choosing among phylogenetic methods.

The relationship between phylogenetic accuracy and congruence between data partitions collected from the same taxa was explored for mitochondrial DNA sequences from two well-supported vertebrate phylogenies. An iterative procedure was adopted whereby accuracy, phylogenetic signal, and congruence were measured before and after modifying a simple reconstruction model, equally weighted parsimony. These modifications included transversion parsimony, successive weighting, and six-parameter parsimony. For the data partitions examined, there is a generally positive relationship between congruence and phylogenetic accuracy. If congruence increased without decreasing resolution or phylogenetic signal, this increased congruence was a good predictor of accuracy. If congruence increased as a result of poor resolution, the degree of congruence was not a good predictor of accuracy. For all sets of data partitions, six-parameter parsimony methods show a consistently positive relationship between congruence and accuracy. Unlike successive weighting, six-parameter parsimony methods were not strongly influenced by the starting tree.

Dramatic mitochondrial gene rearrangements in the hermit crab Pagurus longicarpus (Crustacea, anomura).

The entire mitochondrial gene order of the crustacean Pagurus longicarpus was determined by sequencing all but approximately 300 bp of the mitochondrial genome. We report the first major gene rearrangements found in the clade including Crustacea and Insecta. At least eight mitochondrial gene rearrangements have dramatically altered the gene order of the hermit crab P. longicarpus relative to the putatively ancestral crustacean gene order. These include two rearrangements of protein-coding genes, the first reported for any nonchelicerate arthropod. Codon usage and amino acid sequences do not deviate substantially from those reported for other crustaceans. Investigating the phylogenetic distribution of these eight rearrangements will add additional characters to help resolve decapod phylogeny.

GAMETIC INCOMPATIBILITY BETWEEN SPECIES OF THE SEA URCHIN ECHINOMETRA ON THE TWO SIDES OF THE ISTHMUS OF PANAMA.

The Pliocene rise of the Central American Isthmus has resulted in numerous "geminate pairs," i.e., closely related species, one on each coast. Such species pairs can provide information on the evolution of isolating mechanisms in allopatry and on the relationship between genetic divergence and reproductive isolation in populations separated at a known time. The sea urchin genus Echinometra has one species, E. vanbrunti, in the eastern Pacific, and two, E. lucunter and E. viridis, in the Caribbean. E. viridis is morphologically distinct from the other two species, leading to the conclusion that E. lucunter and E. vanbrunti constitute a geminate pair. Allozyme data, on the other hand, place the speciation event of the two currently sympatric species after the rise of the Isthmus. We report fertilization experiments between the gametes of the three species performed to determine degree of reproductive isolation. Crosses between E. viridis and E. vanbrunti produce rates of fertilization almost equal to those manifested in homogamic crosses. Sperm of E. lucunter can fertilize eggs of the other two species, but few of its eggs permit fertilization by heterospecific sperm. Contrary to the predictions of the "speciation by reinforcement" hypothesis, degree of incompatibility between the allopatric E. lucunter and E. vanbrunti is higher than between the sympatric E. lucunter and E. viridis. Despite the incomplete and unidirectional nature of their gametic isolation, E. lucunter and E. viridis maintain their genetic integrities. Consideration of the likely phylogenetic relationships between the three species suggests that incompatibility of E. lucunter eggs with heterospecific sperm has evolved in the last 3.5 million years, after the rise of the Isthmus. There is no correlation between genetic divergence and strength of reproductive isolation, either within Echinometra, or among the geminate species pairs of echinoids that have been studied to date. Because recognition between echinoid gametes depends on the chemical compositions of a sperm protein and an egg glycoprotein, the appearance of gametic isolation would require only the fixation of a few mutations in each population on either side of a geographic barrier and could be independent of any other kind of genetic divergence. Thus, in animals with external fertilization, speciation need not be accompanied by major genomic reorganization.

MOLECULAR AND GEOLOGIC EVIDENCE OF SHARED HISTORY BETWEEN HERMIT CRABS AND THE SYMBIOTIC GENUS HYDRACTINIA.

The paleobiogeographic histories of three North Atlantic hermit crab lineages were compared with a single-copy DNA-DNA hybridization phylogeny of their symbiotic hydroid genus Hydractinia to test hypotheses of shared history between these host and symbiont lineages. A survey of the geologic literature suggests that two vicariance events in the Quaternary are responsible for existing range disjunctions of the host hermit crab lineages. The Hydractinia phylogeny revealed two distinct clades, one with a primarily northern and the other with a primarily southern distribution. In two of three cases, hydroids associated with closely related hermits on both sides of the range disjunction appear as sister taxa in the phylogeny. A linear scaling between a measure of hydroid sequence divergence and independent geologic estimates of the timing of the vicariant events believed to have established the hermit crab range disjunctions is consistent with the claim of temporal coincidence of cladogenic and vicariance events. These findings provide evidence for shared history of symbiotic associations in two of the three cases.

Reconstructing ancestral character states: a critical reappraisal.

Using parsimony to reconstruct ancestral character states on a phylogenetic tree has become a popular method for testing ecological and evolutionary hypotheses. Despite its popularity, the assumptions and uncertainties of reconstructing the ancestral states of a single character have received less attention than the much less challenging endeavor of reconstructing phylogenetic trees from many characters. Recent research suggests that parsimony reconstructions are often sensitive to violations of the almost universal assumption of equal probabilities of gains and losses. In addition, maximum likelihood has been developed as an alternative to parsimony reconstruction, and has also revealed a surprising amount of uncertainty in ancestral reconstructions.

BEST-FIT MAXIMUM-LIKELIHOOD MODELS FOR PHYLOGENETIC INFERENCE: EMPIRICAL TESTS WITH KNOWN PHYLOGENIES.

Despite the proliferation of increasingly sophisticated models of DNA sequence evolution, choosing among models remains a major problem in phylogenetic reconstruction. The choice of appropriate models is thought to be especially important when there is large variation among branch lengths. We evaluated the ability of nested models to reconstruct experimentally generated, known phylogenies of bacteriophage T7 as we varied the terminal branch lengths. Then, for each phylogeny we determined the best-fit model by progressively adding parameters to simpler models. We found that in several cases the choice of best-fit model was affected by the parameter addition sequence. In terms of phylogenetic performance, there was little difference between models when the ratio of short: long terminal branches was 1:3 or less. However, under conditions of extreme terminal branch-length variation, there were not only dramatic differences among models, but best-fit models were always among the best at overcoming long-branch attraction. The performance of minimum-evolution-distance methods was generally lower than that of discrete maximum-likelihood methods, even if maximum-likelihood methods were used to generate distance matrices. Correcting for among-site rate variation was especially important for overcoming long-branch attraction. The generality of our conclusions is supported by earlier simulation studies and by a preliminary analysis of mitochondrial and nuclear sequences from a well-supported four-taxon amniote phylogeny.

Combining data in phylogenetic analysis.

Systematists have access to multiple sources of character information in phylogenetic analysis. For example, it is not unusual to have nucleotide sequences from several different genes, or to have molecular and morphological data. How should diverse data be analyzed in phylogenetic analysis? Several methods have been proposed for the treatment of partitioned data: the total evidence, separate analysis, and conditional combination approaches. Here, we review some of the advantages and disadvantages of the different approaches, with special concentration on which methods help us to discern the evolutionary process and provide the most accurate estimates of phylogeny.

Evolution of king crabs from hermit crab ancestors.

King crabs (Family Lithodidae) are among the world's largest arthropods, having a crab-like morphology and a strongly calcified exoskeleton. The hermit crabs, by contrast, have depended on gastropod shells for protection for over 150 million years. Shell-living has constrained the morphological evolution of hermit crabs by requiring a decalcified asymmetrical abdomen capable of coiling into gastropod shells and by preventing crabs from growing past the size of the largest available shells. Whereas reduction in shell-living and acquisition of a crab-like morphology (carcinization) has taken place independently in several hermit crab lineages, and most dramatically in king crabs, the rate at which this process has occurred was entirely unknown. We present molecular evidence that king crabs are not only descended from hermit crabs, but are nested within the hermit crab genus Pagurus. We estimate that loss of the shell-living habit and the complete carcinization of king crabs has taken between 13 and 25 million years.

Class-level relationships in the phylum Cnidaria: molecular and morphological evidence.

The evolutionary history of cnidarian life cycles has been debated since the 1880s, with different hypotheses favored even by current textbooks. Contributing to the disagreement is the fact that the systematic relationships of the four cnidarian classes have received relatively little examination using modern systematic methods. Here we present analyses of class-level relationships based on 18S ribosomal DNA (rDNA) sequence, mitochondrial 16S rDNA sequence, mitochondrial genome structure, and morphological characters. DNA sequences were aligned using a repeatable parsimony-based approach incorporating a range of alignment parameters. Analyses of individual data sets and of all data combined are unanimous in grouping the classes possessing a medusa stage, leaving the holobenthic Anthozoa basal within the phylum.