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Novel fossil discoveries have contributed to our understanding of the evolutionary appearance of parasitism in flatworms. Furthermore, genetic analyses with greater coverage have shifted our views on the coevolution of parasitic flatworms and their hosts. The putative record of parasitic flatworms is consistent with extant host associations and so can be used to put constraints on the evolutionary origin of the parasites themselves. The future lies in new molecular clock analyses combined with additional discoveries of exceptionally preserved flatworms associated with hosts and coprolites. Besides direct evidence, the host fossil record and biogeography have the potential to constrain their evolutionary history, albeit with caution needed to avoid circularity, and a need for calibrations to be implemented in the most conservative way. This might result in imprecise, but accurate divergence estimates for the evolution of parasitic flatworms.

Current evidence demonstrates considerable variability in cardiac arrest survival in and out of hospital and, therefore, substantial opportunity to save many more lives.1–3 The Formula for Survival4 postulates that optimal survival from cardiac arrest requires high-quality science, education of lay providers and healthcare professionals, and a well-functioning Chain of Survival5 (implementation). The Education, Implementation, and Teams (EIT) Task Force of the International Liaison Committee on Resuscitation (ILCOR) set out to define the key PICO (population, intervention, comparator, outcome) questions related to resuscitation education (including teamwork skills) and systems-level implementation that would be reviewed by 2015. The selection of questions was supported through the use of an online anonymous task force member–only voting process where the results were considered in the ultimate consensus decisions of the task force. Topics from the 2010 evidence review process were scrutinized for relevance, the potential to improve outcomes, and the likelihood of new evidence being published since 2010. Finally, PICO questions for which the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) process was not as well developed at the time of PICO selection were deferred until at least after the 2015 cycle. We planned to reduce the total number of PICO questions reviewed to provide more in-depth and evidence-based reviews of the included questions. New topics were determined on the basis of the evolving literature and changes in resuscitation practice. Input on the selection of PICO questions was sought from the general public through the ILCOR website and from ILCOR member resuscitation councils through their council chairs and individual task force members. The EIT Task Force performed detailed systematic reviews based on the recommendations of the Institute of Medicine of the National Academies6 and using the methodological approach proposed by the …

Background Madagascar’s rain forests are characterized by extreme and uneven patterns of species richness and endemicity, the biogeographic and evolutionary origins of which are poorly understood. Methods Here we use a time-calibrated phylogeny of a dominant group of trees in Madagascar’s eastern rain forests, Canarium, and related Burseraceae (Canarieae), to test biogeographic hypotheses regarding the origin and radiation of the flora of this unique biome. Results Our findings strongly support the monophyly of Malagasy Canarium, suggesting that this clade represents a previously undocumented in situ radiation. Contrary to expectations of dispersal from Africa during the Oligocene, concurrent with the formation of Madagascar’s rain forest biome, our analyses support a late Miocene origin for Malagasy Canarium, probably by long distance dispersal from Southeast Asia. Discussion Our study illustrates the importance of considering long distance dispersal as a viable explanation for clades with pantropical distributions diversifying subsequent to the Oligocene, and it highlights the formation of the Indo-Australian Archipelago and associated fast-moving equatorial surface currents, suggesting an under-appreciated evolutionary link among tropical centers of endemism. Conclusions We postulate that the relatively recent establishment and radiation of Canarium in Madagascar may have been facilitated by the highly stochastic climates associated with these forest ecosystems.

Fossils provide the principal basis for temporal calibrations, which are critical to the accuracy of divergence dating analyses. Translating fossil data into minimum and maximum bounds for calibrations is the most important, and often least appreciated, step of divergence dating. Properly justified calibrations require the synthesis of phylogenetic, paleontological, and geological evidence and can be difficult for non- specialists to formulate. The dynamic nature of the fossil record (e.g., new discoveries, taxonomic revisions, updates of global or local stratigraphy) requires that calibration data be updated continually lest they become obsolete. Here, we announce the Fossil Calibration Database (http://fossilcalibrations.org), a new open- access resource providing vetted fossil calibrations to the scientific community. Calibrations accessioned into this database are based on individual fossil specimens and follow best practices for phylogenetic justification and geochronological constraint. The associated Fossil Calibration Series, a calibration-themed publication series at Palaeontologia Electronica, will serve as one key pipeline for peer-reviewed calibrations to enter the database.

The timing of divergences among metazoan lineages is integral to understanding the processes of animal evolution, placing the biological events of species divergences into the correct geological timeframe. Recent fossil discoveries and molecular clock dating studies have suggested a divergence of bilaterian phyla100 million years before the Cambrian, when the first definite crown-bilaterian fossils occur. Most previous molecular clock dating studies, however, have suffered from limited data and biases in methodologies, and virtually all have failed to acknowledge the large uncertainties associated with the fossil record of early animals, leading to inconsistent estimates among studies. Here we use an unprecedented amount of molecular data, combined with four fossil calibration strategies (reflecting disparate and controversial interpretations of the metazoan fossil record) to obtain Bayesian estimates of metazoan divergence times. Our results indicate that the uncertain nature of ancient fossils and violations of the molecular clock impose a limit on the precision that can be achieved in estimates of ancient molecular timescales. For example, although we can assert that crown Metazoa originated during the Cryogenian (with most crown-bilaterian phyla diversifying during the Ediacaran), it is not possible with current data to pinpoint the divergence events with sufficient accuracy to test for correlations between geological and biological events in the history of animals. Although a Cryogenian origin of crown Metazoa agrees with current geological interpretations, the divergence dates of the bilaterians remain controversial. Thus, attempts to build evolutionary narratives of early animal evolution based on molecular clock timescales appear to be premature.

The molecular clock is the only viable means of establishing an accurate timescale for Life on Earth, but it remains reliant on a capricious fossil record for calibration. ‘Tip-dating’ promises a conceptual advance, integrating fossil species among their living relatives using molecular/morphological datasets and evolutionary models. Fossil species of known age establish calibration directly, and their phylogenetic uncertainty is accommodated through the co-estimation of time and topology. However, challenges remain, including a dearth of effective models of morphological evolution, rate correlation, the nonrandom nature of missing characters in fossil data, and, most importantly, accommodating uncertainty in fossil age. We show uncertainty in fossil-dating propagates to divergence-time estimates, yielding estimates that are older and less precise than those based on traditional node calibration. Ultimately, node and tip calibrations are not mutually incompatible and may be integrated to achieve more accurate and precise evolutionary timescales. Establishing an evolutionary timescale for Life on Earth has long been a fundamental goal of evolutionary biology, providing the framework for inferring modes and rates of molecular and phenotypic evolution, as well as a means of associating intrinsic evolutionary change to extrinsic causal factors. This endeavor was originally the domain of paleontologists, but it is now widely accepted that fossil data alone are insufficient because of the incompleteness of the fossil record [1]. Molecular clock dating methodology can be used to establish an evolutionary timescale by calculating the molecular distance between species, and by estimating absolute molecular evolutionary rates based on the oldest fossil evidence for the antiquity of the living lineages [2]. This powerful combination of molecular and paleontological data sees through the gaps in the fossil record, providing the only viable means of establishing an accurate evolutionary timescale. Molecular clock methodology has been developed to accommodate tree-wide substitution rate

Exceptionally preserved fossils provide major insights into the evolutionary history of life. Microbial activity is thought to play a pivotal role in both the decay of organisms and the preservation of soft tissue in the fossil record, though this has been the subject of very little experimental investigation. To remedy this, we undertook an experimental study of the decay of the brine shrimp Artemia, examining the roles of autolysis, microbial activity, oxygen diffusion and reducing conditions. Our findings indicate that endogenous gut bacteria are the main factor controlling decay. Following gut wall rupture, but prior to cuticle failure, gut-derived microbes spread into the body cavity, consuming tissues and forming biofilms capable of mediating authigenic mineralization, that pseudomorph tissues and structures such as limbs and the haemocoel. These observations explain patterns observed in exceptionally preserved fossil arthropods. For example, guts are preserved relatively frequently, while preservation of other internal anatomy is rare. They also suggest that gut-derived microbes play a key role in the preservation of internal anatomy and that differential preservation between exceptional deposits might be because of factors that control autolysis and microbial activity. The findings also suggest that the evolution of a through gut and its bacterial microflora increased the potential for exceptional fossil preservation in bilaterians, providing one explanation for the extreme rarity of internal preservation in those animals that lack a through gut.

Hospital care on weekends has been associated with delays in care, reduced quality, and poor clinical outcomes. The purpose of this study was to evaluate the impact of a weekend hospital intervention on processes of care and clinical outcomes. The multifaceted intervention included expanded weekend diagnostic services, improved weekend discharge processes, and increased physician and care management services on weekends. This was an interrupted time series observational study of adult non-obstetric patients hospitalized at a single academic medical center between January 2011 and January 2014. The study included 18 months prior to and 19 months following the implementation of the intervention. Data were analyzed using segmented regression analysis with adjustment for confounders. The primary outcome was average length of stay. Secondary outcomes included percent of patients discharged on weekends, 30-day readmission rate, and in-hospital mortality rate. The study included 57,163 hospitalizations. Following implementation of the intervention, average length of stay decreased by 13 % (95 % CI 10–15 %) and continued to decrease by 1 % (95 % CI 1–2 %) per month as compared to the underlying time trend. The proportion of weekend discharges increased by 12 % (95 % CI 2–22 %) at the time of the intervention and continued to increase by 2 % (95 % CI 1–3 %) per month thereafter. The intervention had no impact on readmissions or mortality. During the post-implementation period, the hospital was evacuated and closed for 2 months due to damage from Hurricane Sandy, and a new hospital-wide electronic health record was introduced. The contributions of these events to our findings are not known. We observed a lower inpatient census and found differences in patient characteristics, including higher rates of Medicaid insurance and comorbidities, in the post-Hurricane Sandy period as compared to the pre-Sandy period. The intervention was associated with a reduction in length of stay and an increase in weekend discharges. Our longitudinal study also illuminated the challenges of evaluating the effectiveness of a large-scale intervention in a real-world hospital setting.

The distinctly non-random diversity of organis- mal form manifests itself in discrete clusters of taxa that share a common body plan. As a result, analyses of disparity require a scalable comparative framework. The difficulties of applying geometric morphometrics to disparity analyses of groups with vastly divergent body plans are overcome partly by the use of cladistic characters. Character-based disparity analyses have become increasingly popular, but it is not clear how they are affected by character coding strategies or revi- sions of primary homology statements. Indeed, whether cla- distic and morphometric data capture similar patterns of morphological variation remains a moot point. To address this issue, we employ both cladistic and geometric morpho- metric data in an exploratory study of disparity focussing on caecilian amphibians. Our results show no impact on relative intertaxon distances when different coding strategies for cla- distic characters were used or when revised concepts of homology were considered. In all instances, we found no sta- tistically significant difference between pairwise Euclidean and Procrustes distances, although the strength of the corre- lation among distance matrices varied. This suggests that cla- distic and geometric morphometric data appear to summarize morphological variation in comparable ways. Our results support the use of cladistic data for characteriz- ing organismal disparity.

Dating analyses based on molecular data imply that crown angiosperms existed in the Triassic, long before their undisputed appearance in the fossil record in the Early Cretaceous. Following a re-analysis of the age of angiosperms using updated sequences and fossil calibrations, we use a series of simulations to explore the possibility that the older age estimates are a consequence of (i) major shifts in the rate of sequence evolution near the base of the angiosperms and/or (ii) the representative taxon sampling strategy employed in such studies. We show that both of these factors do tend to yield substantially older age estimates. These analyses do not prove that younger age estimates based on the fossil record are correct, but they do suggest caution in accepting the older age estimates obtained using current relaxed-clock methods. Although we have focused here on the angiosperms, we suspect that these results will shed light on dating discrepancies in other major clades.

Campylobacter is a leading cause of foodborne illness worldwide. It is common in poultry, and human infections are often associated with consumption of contaminated poultry products. One strategy to reduce Campylobacter colonization in poultry is the use of oral probiotics, but this produces variable results, possibly because the probiotics are destroyed in the stomach's acidic environment. Protection (e.g., encapsulation) of isolates may overcome this problem, but there is no assurance that these isolates will have efficacy in the lower gastrointestinal tract. Therefore, screening candidate isolates by directly placing them in the lower intestinal tract via cloacal inoculation may eliminate the time and expense of encapsulating ineffective isolates. Thus, the purpose of this study was to collect bacterial isolates with anti-Campylobacter activity in vitro and evaluate their efficacy in vivo upon either oral or intracloacal administration. Bacterial isolates were collected from healthy birds and were evaluated for efficacy against C. jejuni in vitro. Isolates having generally regarded as safe status and demonstrating in vitro anti-Campylobacter properties were evaluated after oral or intracloacal inoculation into chicks on day 1 (n = 10 birds per isolate per route of administration). On day 7, birds were dosed by oral gavage with a four-strain mixture of wild-type Campylobacter containing at least 1 × 10(7) CFU/ml organisms. On day 14, birds were euthanized and the ceca were collected aseptically for Campylobacter enumeration. When dosed orally, only one isolate had a 1-log reduction in cecal Campylobacter counts, whereas when administered intracloacally, six of these isolates produced a 1- to 3-log reduction in cecal Campylobacter counts in 14-day-old chickens. These results support the strategy of evaluating the efficacy of potential probiotic isolates via cloacal inoculation prior to undergoing the effort of encapsulating isolates for oral administration.

For anyone who has cared for animal embryos, it beggars belief that these squishy cellular aggregates could be fossilised. Hence, with hindsight, it is possible to empathise with palaeontologists who found such fossils and, in their naming of Olivooides, Pseudooides, etc., drew attention to their likeness to animal eggs and embryos but without going so far as to propose such an interpretation. However, in 1994, Zhang Xi-guang and Brian Pratt described microscopic balls of calcium phosphate from Cambrian rocks of China, one or two of which preserved polygonal borders that resembled blastomeres on the surface of an early cleaving animal embryo. In retrospect, these fossils are far from remarkable, some of them may not be fossils at all, and it is not as if anyone ever conceived Cambrian animals as having lacked an embryology. But Zhang Xi-guang and Brian Pratt dared the scientific world, not least their fellow palaeontologists, to believe that the fragile embryonic stages of invertebrate animals could be fossilised, that there was a fossil record of animal embryology, that this record hailed from the interval of time in which animal body plans were first established, and that it had been awaiting discovery in the rocks, for want of looking. The proof of this concept came a few years later, when phosphatised Cambrian fossils from China and Siberia were shown to display indisputable features of animal embryonic morphologies. In the case of Olivooides, a series of developmental stages from cleavage to morphogenesis through hatching and juvenile growth could be tentatively identified; in Markuelia, the coiled-up body of an annulated worm-like animal could be clearly seen within its fertilisation envelope.

Novel fossil discoveries have contributed to our understanding of the evolutionary appearance of parasitism in flatworms. Furthermore, genetic analyses with greater coverage have shifted our views on the coevolution of parasitic flatworms and their hosts. The putative record of parasitic flatworms is consistent with extant host associations and so can be used to put constraints on the evolutionary origin of the parasites themselves. The future lies in new molecular clock analyses combined with additional discoveries of exceptionally preserved flatworms associated with hosts and coprolites. Besides direct evidence, the host fossil record and biogeography have the potential to constrain their evolutionary history, albeit with caution needed to avoid circularity, and a need for calibrations to be implemented in the most conservative way. This might result in imprecise, but accurate divergence estimates for the evolution of parasitic flatworms.

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.

The immune system of neonatal chicks is functionally immature during the first week of life. Researchers have previously demonstrated that the avian humoral response can be increased with probiotics. Although the humoral response provides the chick with an effective mechanism to combat pathogens, sufficient antibody titers are not attained until 7 to 10 d postinfection. However, the innate immune system (i.e., heterophils) can respond much more quickly to pathogens. The objective of this study was to determine whether probiotic bacteria can also upregulate heterophil function. Heterophils were isolated from the peripheral blood of neonatal chickens by using a discontinuous density gradient. Oxidative burst and degranulation are bactericidal mechanisms used by heterophils to kill pathogens and were used in this study as indicators of heterophil function. We found that each of the 10 "generally recognized as safe" probiotic isolates (designated G1 to G11) tested in vitro were capable of increasing (P < 0.05) heterophil oxidative burst and degranulation when compared with unstimulated controls. Bacillus subtilis (G3), Lactococcus lactis lactis (G6), and Lactobacillus acidophilus (G8) isolates were determined to elicit the greatest heterophil response in vitro and were subsequently fed to chicks. Phosphate-buffered saline or 1 of these 3 probiotic isolates (approximately 2.5 x 10(8) cfu/chick; 50 chicks/treatment) resuspended in PBS was administered by oral gavage on the day of hatch. Heterophils were isolated from chicks from each of these 4 treatment groups 24 h posttreatment. Significant increases in heterophil degranulation and oxidative burst were observed with the G3-, G6-, and G8-treated chicks when compared with heterophils isolated from birds with no probiotic treatment. These data suggest that probiotic bacteria can significantly improve heterophil oxidative burst and degranulation in broilers. To our knowledge, this is the first study demonstrating a relationship between probiotics and avian heterophil function.

The role of fossils in dating the tree of life has been misunderstood. Fossils can provide good "minimum" age estimates for branches in the tree, but "maximum" constraints on those ages are poorer. Current debates about which are the "best" fossil dates for calibration move to consideration of the most appropriate constraints on the ages of tree nodes. Because fossil-based dates are constraints, and because molecular evolution is not perfectly clock-like, analysts should use more rather than fewer dates, but there has to be a balance between many genes and few dates versus many dates and few genes. We provide "hard" minimum and "soft" maximum age constraints for 30 divergences among key genome model organisms; these should contribute to better understanding of the dating of the animal tree of life.

Electron transfer is the simplest reaction possible, yet it has a profound impact on the structure and reactivity of organic compounds. These changes allow a new look at some of the fundamental concepts that are used to explain organic chemistry, such as symmetry, aromaticity, and bonding. The results from high-level electronic structure calculations are used to analyze the mechanistic differences in the pericyclic reactions of simple hydrocarbons and their radical cation counterparts. The importance of state symmetry correlation, Jahn-Teller distortions, delocalization, and fractional bonding for the reaction pathways of hydrocarbon radical cations is discussed.

Campylobacter is a leading cause of food-borne illness in the United States. Recent evidence has demonstrated that bacteriocins produced by Bacillus circulans and Paenibacillus polymyxa reduce cecal Campylobacter colonization in broiler chickens infected with Campylobacter jejuni. As Campylobacter coli is the most prevalent Campylobacter isolate recovered in turkeys, the objectives of the present study were to evaluate the efficacy of these bacteriocins against C. coli colonization and their influence on the gastrointestinal architecture of young turkeys. In 3 separate trials, a total of 135 day-of-hatch poults (n = 45/trial) were orally challenged on d 3 with approximately 10(6) cfu of a mixture of 3 C. coli isolates. Immediately before bacteriocin treatment (d 10), cecal Campylobacter concentrations averaged 1.1 x 10(7) cfu/ g of cecal contents (n = 15/trial). On d 10 to 12 posthatch, 2 bacteriocin treatment groups were given free access to feed supplemented with purified, microencapsulated bacteriocins, whereas the positive control treatment group had access to untreated feed (n = 10/treatment group per trial). At the end of the 3-d dosing period, ceca and duodenal loops were collected for analysis. In each of the 3 separate trials, treatment with bacteriocin eliminated detectable ceca Campylobacter concentrations (detection limit, 1 x 10(2) cfu/g of cecal contents) vs. controls (1.0 x 106 cfu of Campylobacter/g of cecal contents). Duodenum crypt depth and goblet cell numbers were also reduced in turkeys treated with either bacteriocin vs. controls (P < 0.05). The dynamic reduction in crypt depth and goblet cell density in turkeys dosed with bacteriocin may provide clues to how bacteriocins inhibit enteric Campylobacter.

The estimation of ever larger phylogenies requires consideration of alternative inference strategies, including divide-and-conquer approaches that decompose the global inference problem to a set of smaller, more manageable component problems. A prominent locus of research in this area is the development of supertree methods, which estimate a composite tree by combining a set of partially overlapping component topologies. Although promising, the use of component tree topologies as the primary data dissociates supertrees from complexities within the underling character data and complicates the evaluation of phylogenetic uncertainty. We address these issues by exploring three approaches that variously incorporate nonparametric bootstrapping into a common supertree estimation algorithm (matrix representation with parsimony, although any algorithm might be used), including bootstrap-weighting, source-tree bootstrapping, and hierarchical bootstrapping. We illustrate these procedures by means of hypothetical and empirical examples. Our preliminary experiments suggest that these methods have the potential to improve the correspondence of supertree estimates to those derived from simultaneous analysis of the combined data and to allow uncertainty in supertree topologies to be quantified. The ability to increase the transparency of supertrees to the underlying character data has several practical implications and sheds new light on an old debate. These methods have been implemented in the freely available program, tREeBOOT.

Fossilized embryos from the late Neoproterozoic and earliest Phanerozoic have caused much excitement because they preserve the earliest stages of embryology of animals that represent the initial diversification of metazoans. However, the potential of this material has not been fully realized because of reliance on traditional, non-destructive methods that allow analysis of exposed surfaces only, and destructive methods that preserve only a single two-dimensional view of the interior of the specimen. Here, we have applied synchrotron-radiation X-ray tomographic microscopy (SRXTM), obtaining complete three-dimensional recordings at submicrometre resolution. The embryos are preserved by early diagenetic impregnation and encrustation with calcium phosphate, and differences in X-ray attenuation provide information about the distribution of these two diagenetic phases. Three-dimensional visualization of blastomere arrangement and diagenetic cement in cleavage embryos resolves outstanding questions about their nature, including the identity of the columnar blastomeres. The anterior and posterior anatomy of embryos of the bilaterian worm-like Markuelia confirms its position as a scalidophoran, providing new insights into body-plan assembly among constituent phyla. The structure of the developing germ band in another bilaterian, Pseudooides, indicates a unique mode of germ-band development. SRXTM provides a method of non-invasive analysis that rivals the resolution achieved even by destructive methods, probing the very limits of fossilization and providing insight into embryology during the emergence of metazoan phyla.

In several areas of southern Sweden, limestone nodules, locally called Orsten occur within bituminous alum shales. These shales and nodules were deposited under dysoxic conditions at the bottom of what was most likely a shallow sea during the late Middle to Upper Cambrian (ca. 500 million years ago). Subsequently, the name 'Orsten' has been referred to particular, mainly arthropod, fossils from such nodules, and, in a wider sense, to the specific type of preservation of minute fossil through secondarily phosphatization. This preservation is exceptional in yielding uncompacted and diagenetically undeformed three-dimensional fossils. 'Orsten'-type preservation resulted from incrustation of a thin external layer and also by impregnation by calcium phosphate and, therefore, mineralization of the surface of the former animals during early diagenesis. Primarily, this type of preservation seems to have affected only cuticle-bearing metazoans such as cycloneuralian nemathelminths and arthropods. 'Orsten' preservation in this sense seems to be limited by size, in having yielded no partial or complete animals larger than 2 mm. On the other end of the scale, even larvae 100 μm long are preserved, often more complete than larger specimens, and details such as setules and pores smaller than 1 μm can be observed. Fossils preserved in such a manner are almost exclusively hollow carcasses, but can be filled secondarily; less common are completely phosphatized compact specimens. The high quality of preservation makes the Swedish 'Orsten' a typical Konservat-Lagerstätte. Yet, its special type of preservation is more widespread in time and geographical distribution than assumed initially, and the origin of the phosphate is not necessarily restricted just to one source. Subsequent to the first discoveries of limb fragments of Cambrian arthropods in 1975, animals in this special preservational type have been discovered in several continents and across a broad stratigraphic range including even Proterozoic strata. The latter have yielded early cleavage and metazoan embryonic stages, expanding knowledge on the preservational capacities of the 'Orsten'. Here, we report the recent status of our research on the 'Orsten' and give perspectives for future exploration on a worldwide scale, particularly in light of a recently formed international research group named Center of Orsten Research and Exploration (C.O.R.E.). © 2006.

The cactus life-form is cited as an example of a tight relationship between organism form and function: a succulent, long-lived, photosynthetic stem allows cacti to survive long periods of drought while maintaining a positive tissue water status. Pereskia (Cactaceae) comprises 17 species of leafy shrubs and trees that are thought to represent the original cactus condition. Recent phylogenetic work has shown that there are two separate clades of Pereskia species, which are basal and paraphyletic with respect to the rest of the cacti. We selected seven Pereskia species, representing both clades, and characterized their water relations by measuring a suite of physiological traits in wild populations. Additionally, we estimated basic climate parameters from collection localities for all 17 Pereskia species. Extant Pereskia species exhibit ecological water use patterns that are very similar to those of the leafless, stem-succulent cacti. Ancestral trait reconstruction for the physiological and environmental data provides a preliminary assessment of the ecology and water relations of the earliest cacti and suggests that several key elements of the cactus ecological niche were established before the evolution of the cactus life-form. We interpret these ecological traits as potentially important drivers of evolutionary innovation in the cacti.

The stratigraphically earliest and the most primitive examples of vertebrate skeletal mineralization belong to lineages that are entirely extinct. Therefore, palaeontology offers a singular opportunity to address the patterns and mechanisms of evolution in the vertebrate mineralized skeleton. We test the two leading hypotheses for the emergence of the four skeletal tissue types (bone, dentine, enamel, cartilage) that define the present state of skeletal tissue diversity in vertebrates. Although primitive vertebrate skeletons demonstrate a broad range of tissues that are difficult to classify, the first hypothesis maintains that the four skeletal tissue types emerged early in vertebrate phylogeny and that the full spectrum of vertebrate skeletal tissue diversity is explained by the traditional classification system. The opposing hypothesis suggests that the early evolution of the mineralized vertebrate skeleton was a time of plasticity and that the four tissue types did not emerge until later. On the basis of a considerable, and expanding, palaeontological dataset, we track the stratigraphic and phylogenetic histories of vertebrate skeletal tissues. With a cladistic perspective, we present findings that differ substantially from long-standing models of tissue evolution. Despite a greater diversity of skeletal tissues early in vertebrate phylogeny, our synthesis finds that bone, dentine, enamel and cartilage do appear to account for the full extent of this variation and do appear to be fundamentally distinct from their first inceptions, although why a higher diversity of tissue structural grades exists within these types early in vertebrate phylogeny is a question that remains to be addressed. Citing recent evidence that presents a correlation between duplication events in secretory calcium-binding phosphoproteins (SCPPs) and the structural complexity of mineralized tissues, we suggest that the high diversity of skeletal tissues early in vertebrate phylogeny may result from a low diversity of SCPPs and a corresponding lack of constraints on the mineralization of these tissues.

A reversed-phase liquid chromatographic (LC) method is presented for the analysis of N-methyl carbamate pesticide residues and piperonyl butoxide in eggs at levels as low as 2 gkg (ppb). The study was undertaken to provide data for dietary exposure estimates used in risk analysis. The method uses an acetonitrile extraction followed by liquidliquid partitioning and normal-phase aminopropyl solid-phase extraction column cleanup. Determination of residues is by reversed-phase LC with an inline postcolumn reaction followed by fluorescence detection. The average recoveries of 21 fortified (most at 2.0 and 20.0 ppb) N-methyl carbamate pesticide residues and the carbamate metabolite 1-naphthol from eggs ranged from 70 to 107%. Recoveries of the pesticide synergist piperonyl butoxide ranged from 63 to 106%. Single-comb White Leghorn hens were treated with the carbamate carbaryl, and the eggs subsequently produced were analyzed for carbaryl and 1-naphthol residues.

The histological composition of the galeaspid cephalothoracic skeleton has been much debated: here we attempt to resolve this through the analysis of well-preserved remains of galeaspids from Yunna...

The classification of fossils has long been controversial. Should traditional taxonomic concepts be expanded to encompass plesiomorphic extinct relatives that exhibit subsets of essential defining characters in so many shades of gray? Should rank taxa, established on extant taxa alone, and in a pre-Darwinian, pre-Hennigian framework, remain restricted to their living constituents and integral extinct relatives? Or should these taxonomic concepts be restricted to a membership exhibiting a defining suite of essential characters, regardless of whether they are extant or extinct?

M.B. FARNELL, A.M. DONOGHUE, K. COLE, I. REYES-HERRERA, P.J. BLORE AND D.J. DONOGHUE. 2005. Aims: This study evaluated the relationship between Campylobacter susceptibility and enteric fluoroquinolone concentrations in chickens treated with different doses of enrofloxacin. Methods and Results: All chickens were challenged with seven fluoroquinolone sensitive Campylobacter jejuni (6AE6 · 10 6 CFU per bird) at 2 weeks posthatch. At 26 days of age chickens were treated with 0 (n ¼ 29 birds), 25 mg ml )1 enrofloxacin (Baytril� , Bayer Corp., Shawnee Mission, KS, USA) for 3 days (n ¼ 45 birds) or 50 mg ml )1 enrofloxacin for 7 days (n ¼ 65 birds) in the drinking water. The crop, upper ileum, lower ileum, ceca and colon contents were collected from both enrofloxacin treatment groups (n ¼ 5 birds per day per treatment group) and nonmedicated controls. The minimum inhibitory concentration (MIC) of ciprofloxacin for Campylobacter increased for isolates from both treatment groups within the first day of dosing and the daily average ranged from 1AE 4t o 6AE5 l gm l )1 throughout the study. Although enteric fluoroquinolone concentrations were higher (P

At a time when historical biogeography appears to be again expanding its scope after a period of focusing primarily on discerning area relationships using cladograms, new inference methods are needed to bring more kinds of data to bear on questions about the geographic history of lineages. Here we describe a likelihood framework for inferring the evolution of geographic range on phylogenies that models lineage dispersal and local extinction in a set of discrete areas as stochastic events in continuous time. Unlike existing methods for estimating ancestral areas, such as dispersal-vicariance analysis, this approach incorporates information on the timing of both lineage divergences and the availability of connections between areas (dispersal routes). Monte Carlo methods are used to estimate branch-specific transition probabilities for geographic ranges, enabling the likelihood of the data (observed species distributions) to be evaluated for a given phylogeny and parameterized paleogeographic model. We demonstrate how the method can be used to address two biogeographic questions: What were the ancestral geographic ranges on a phylogenetic tree? How were those ancestral ranges affected by speciation and inherited by the daughter lineages at cladogenesis events? For illustration we use hypothetical examples and an analysis of a Northern Hemisphere plant clade (Cercis), comparing and contrasting inferences to those obtained from dispersal-vicariance analysis. Although the particular model we implement is somewhat simplistic, the framework itself is flexible and could readily be modified to incorporate additional sources of information and also be extended to address other aspects of historical biogeography.

Ectopic expression of fibroblast growth factor receptor 3 (FGFR3) associated with t(4;14) has been implicated in the pathogenesis of human multiple myeloma. Some t(4;14) patients have activating mutations of FGFR3, of which a minority are K650E (thanatophoric dysplasia type II [TDII]). To investigate the role of autophosphorylated tyrosine residues in FGFR3 signal transduction and transformation, we characterized a series of FGFR3 TDII mutants with single or multiple Y--F substitutions. Phenylalanine substitution of Y760, essential for phospholipase Cgamma (PLCgamma) binding and activation, significantly attenuated FGFR3 TDII-mediated PLCgamma activation, as well as transformation in Ba/F3 cells and a murine bone marrow transplant leukemia model. In contrast, single substitution of Y577, Y724, or Y770 had minimal to moderate effects on TDII-dependent transformation. Substitution of all 4 non-activation loop tyrosine residues significantly attenuated, but did not abolish, TDII transforming activity. Similar observations were obtained in the context of a constitutively activated fusion TEL-FGFR3 associated with t(4;12)(p16;p13) peripheral T-cell lymphomas. Moreover, 2 independent EmuSR-FGFR3 TDII transgenic mouse lines developed a pro-B-cell lymphoma, and PLCgamma was highly activated in primary lymphoma cells as assessed by tyrosine phosphorylation. These data indicate that engagement of multiple signaling pathways, including PLCgamma-dependent and PLCgamma-independent pathways, is required for full hematopoietic transformation by constitutively activated FGFR3 mutants.

Vertebrate evolution has been punctuated by three episodes of widespread gene or genome duplication, which have been linked with the origin of vertebrates, gnathostomes and teleosts, respectively. These three events coincide with bursts of character acquisition and increases in phenotypic complexity, and many researchers have suggested a causal relationship between the two. However, this pattern is derived from data for living taxa only; we argue here that, when fossils are taken into account, bursts of character acquisition disappear and gen(om)e duplication in vertebrate phylogeny can no longer be correlated with the origin of body plans. If patterns of character acquisition or morphological gaps between higher taxa are a reflection of phenotypic complexity, then more inclusive data sets incorporating fossil taxa provide no support for hypotheses linking gen(om)e duplications and the evolution of complexity in vertebrates.

Improvements in our understanding of green plant phylogeny are casting new light on the connection between character evolution and diversification. The repeated discovery of paraphyly has helped disentangle what once appeared to be phylogenetically coincident character changes, but this has also highlighted the existence of sequences of character change, no one element of which can cleanly be identified as the “key innovation” responsible for shifting diversification rate. In effect, the cause becomes distributed across a nested series of nodes in the tree. Many of the most conspicuous plant “innovations” (such as macrophyllous leaves) are underlain by earlier, more subtle shifts in development (such as overtopping growth), which appear to have enabled the exploration of a greater range of morphological designs. Often it appears that these underlying changes have been brought about at the level of cell interactions within meristems, highlighting the need for developmental models and experiments focused at this level. The standard practice of attempting to identify correlations between recurrent character change (such as the tree growth habit) and clade diversity is complicated by the observation that the “same” trait may be constructed quite differently in different lineages (e.g., different forms of cambial activity), with some solutions imposing more architectural limitations than others. These thoughts highlight the need for a more nuanced view, which has implications for comparative methods. They also bear on issues central to Stephen Jay Gould's vision of macroevolution, including exaptation and evolutionary recurrence in relation to constraint and the repeatability of evolution.

Flower symmetry is of special interest in understanding the evolution and ecology of angiosperms. Evidence from the Antirrhineae (within the lamiid clade of Asteridae) indicates that several TCP gene family transcription factors, especially CYCLOIDEA (CYC), play a role in specifying dorsal identity in the corolla and androecium of monosymmetric (bilateral) flowers. We examine the evolution of this gene family in Dipsacales, representing the campanulid clade of Asteridae, in relation to evolutionary shifts in floral symmetry and stamen abortion. We identify three major forms of CYC-like genes in Dipsacales. We identify the position of additional gene duplications by comparing each of the three gene trees to a well-supported Dipsacales phylogeny. We infer duplications in two of the major gene lineages along the line leading to the Caprifoliaceae correlated with the origin of monosymmetric flowers. There are no duplications or losses associated with major shifts in stamen number. However, there are several additional duplications within the Caprifoliaceae, especially in the Morinaceae, possibly related to calyx monosymmetry and/or stamen reduction. Within the protein-coding sequences, we identify a new conserved region—the ECE region—that appears to be present across known angiosperm sequences. There are major changes in length and in the presence or absence of the ECE region in the Dipsacales, indicating changes in gene function. These studies expand our understanding of the evolution of CYC-like genes in angiosperms and provide a new system for studying the role of this gene family in determining floral form.

We investigated Viburnum phylogeny using separate and combined analyses of DNA sequence data from two chloroplast and three nuclear loci. Separate analyses of nuclear and chloroplast data sets resulted in gene trees that were generally congruent with one another and with trees from two previous analyses. Our gene trees do differ in the position of section Pseudotinus, as well as in species relationships within sections Pseudotinus and Lentago. However, tests for incongruence indicate that differences between the nuclear and chloroplast data are not significant. Furthermore, gene trees from combined analyses were highly similar to those found in separate analyses, suggesting that these localized differences do not affect other parts of the tree. Our analyses provide convincing support for numerous relationships, although there is still uncertainty at the base of the tree. To facilitate future study, we propose informal names for 12 well-supported species groups, as well as for several higher-level clades. We also discuss the biogeographic implications of our phylogeny, focusing on repeated, although apparently temporally incongruent, patterns of disjunction between the Old and New Worlds.

Japanese quail provide an advantageous avian model for assessing long-term biological consequences of endocrine disrupting chemicals (EDCs). These studies examined route of exposure and vulnerability to biological impact of EDCs over the life cycle in a precocial avian model, the Japanese quail. Embryonic exposure occurs with maternal deposition and methoxychlor (MXC) accumulated with maternal exposure. Egg injections of MXC or estradiol at selected stages of development impacted hypothalamic neuroendocrine systems in hatchlings and affected sexual maturation, with evidence for long-term effects on neurotransmitters and male behavior. Two-generation dietary studies were conducted to examine transgenerational effects of EDCs. Adult quail (P1) were exposed to dietary MXC (0, 0.5 and 5 ppm), with continued exposure in their offspring (F1), and control diet for all F2 chicks. Toxicological end points, including fertility, hatching success, and 14-day viability were unaffected. F1 and F2 male offspring from MXC-treated pairs MXC had impaired mating behavior and altered plasma hormones. These studies confirm neuroendocrine and behavioral measures as reliable indices of exposure to an estrogenic EDC. Moreover, maternal deposition remains a primary route of EDC exposure, with potential deleterious consequences for field birds, especially precocial species that appear to be particularly sensitive to embryonic EDC exposure.

The hypothesis that conodonts are vertebrates rests solely on evidence of soft tissue anatomy. This has been corroborated by microstructural, topological and develop- mental evidence of homology between conodont and verteb- rate hard tissues. However, these conclusions have been reached on the basis of evidence from highly derived eucono- dont taxa and the degree to which they are representative of plesiomorphic euconodonts remains an open question. Fur- thermore, the range of variation in tissue types comprising the euconodont basal body has been used to establish a hypo- thesis of developmental plasticity early in the phylogeny of the clade, and a model of diminishing potentiality in the evo- lution of development systems. The microstructural fabrics of the basal tissues of the earliest euconodonts (presumed to be the most plesiomorphic) are examined to test these two hypo- theses. It is found that the range of microstructural variation observed hitherto was already apparent among plesiomorphic euconodonts. Thus, established histological data are represen- tative of the most plesiomorphic euconodonts. However, although there is evidence of a range in microstructural fab- rics, these are compatible with the dentine tissue system alone, and the degree of variation is compatible with that seen in clades of comparable diversity.

Fossil data have been interpreted as indicating that Late Cretaceous tropical forests were open and dry adapted and that mod- ern closed-canopy rain forest did not originate until after the Cretaceous-Tertiary (K/T) boundary. However, some mid-Cretaceous leaf floras have been interpreted as rain forest. Molecular divergence- time estimates within the clade Malpighiales, which constitute a large percentage of species in the shaded, shrub, and small tree layer in tropical rain forests worldwide, provide new tests of these hypotheses. We estimate that all 28 major lineages (i.e., traditionally recognized families) within this clade originated in tropical rain forest well before the Tertiary, mostly during the Albian and Cenomanian (112-94 Ma). Their rapid rise in the mid-Cretaceous may have resulted from the origin of adaptations to survive and reproduce under a closed forest canopy. This pattern may also be paralleled by other similarly diverse lineages and supports fossil indications that closed-canopy tropical rain forests existed well before the K/T boundary. This case illustrates that dated phylogenies can provide an important new source of ev- idence bearing on the timing of major environmental changes, which may be especially useful when fossil evidence is limited or controversial.

Dipsacales is an asterid angiosperm clade of ca. 1100 species, with most of its lineages occupying temperate regions of the Northern Hemisphere. A recent phylogenetic analysis based on 7593 nucleotides of chloroplast DNA recovered a well-resolved and strongly supported phylogenetic hypothesis, which we use here to estimate divergence times within the group. A molecular clock is strongly rejected, regardless of data partition. We used recently proposed methods that relax the assumption of rate constancy among lineages (local clocks, nonparametric rate smoothing, penalized likelihood, and Bayesian relaxed clock) to estimate the ages of major lineages. Age estimates for Dipsacales varied widely among markers and codon positions, and depended on the fossils used for calibration and method of analysis. Some methods yielded dates for the Dipsacales diversification that appear to be too old (prior to the presumed 125 my [million years] age of eudicots), and others suggested ages that are too young based on well-documented Dipsacales fossils. Concordant penalized likelihood and Bayesian studies imply that Dipsacales originated in the Cretaceous, as did its two major lineages, Adoxaceae and Caprifoliaceae. However, diversification of crown Adoxaceae and Caprifoliaceae mainly occurred in the Tertiary, with the origin of major lineages within these clades mainly occurring during the Eocene. Another round of diversification appears to have occurred in the Miocene. Several radiations, such as Valerianaceae in South America and Dipsacaceae around the Mediterranean, are even more recent. This study demonstrates the wide range of divergence times that can be obtained using different methods and data sets, and cautions against reliance on age estimates based on only a single gene or methodology. Despite this variance, significant conclusions can be made about the timing of Dipsacales evolution.

Ecology and historical (phylogeny-based) biogeography have much to offer one another, but exchanges between these fields have been limited. Historical biogeography has become narrowly focused on using phylogenies to discover the history of geological connections among regions. Conversely, ecologists often ignore historical biogeography, even when its input can be crucial. Both historical biogeographers and ecologists have more-or-less abandoned attempts to understand the processes that determine the large-scale distribution of clades. Here, we describe the chasm that has developed between ecology and historical biogeography, some of the important questions that have fallen into it and how it might be bridged. To illustrate the benefits of an integrated approach, we expand on a model that can help explain the latitudinal gradient of species richness.

Recent studies of Northern Hemisphere biogeography have highlighted potentially significant differences between disjunction patterns in plants versus animals. To assess such differences, we compiled a larger sample of relevant plant phylogenies from which disjunction patterns, ancestral areas and directions of movement could be inferred. We considered 66 plant clades with species variously endemic today to eastern Asia (EA), Europe (including southwestern Asia), eastern North America (ENA), and/or western North America (WNA). Within these clades we focused on 100 disjunctions among these major areas, for 33 of which absolute divergence times have also been inferred. Our analyses uphold the view that disjunctions between EA and ENA are exceptionally common in plants, apparently more so than in animals. Compared with animals, we find few disjunctions between EA and WNA, consistent with increased extinction in WNA or failure of some groups to colonize that region. Taken at face value, our data also support the view that many temperate forest plant groups originated and diversified within EA, followed by movement out of Asia at different times, but mostly during the last 30 Myr. This favours Beringia over a North Atlantic land bridge as the primary path between the Old World and the New World. Additional studies are needed, especially to evaluate the impacts of differential extinction on these patterns, to more confidently establish divergence times, and to assess the statistical significance of these findings. Fortunately, many more plant groups show relevant disjunction patterns and could soon be added to such analyses.

DNA sequencing studies of the granule-bound starch synthase gene (GBSSI) indicate the presence of two loci in Viburnum. Gene trees from separate and combined phylogenetic analyses of the GBSSI paralogues are generally congruent with each other and with trees from previous analyses, especially those of Donoghue et al. [Syst. Bot. 29 (2004) 188] based on nuclear ribosomal ITS and chloroplast trnK intron DNA sequences. Specifically, our GBSSI trees confirm (i) the monophyly of some and non-monophyly of other traditionally recognized taxonomic sections, (ii) the presence of three major supra-sectional lineages within Viburnum, and (iii) the resolution of many species relationships within the section-level clades. Analyses of GBSSI also provide greater resolution of relationships within the largest supra-sectional lineage. Relationships at the base of the Viburnum phylogeny remain uncertain; in particular, the position of the root, relationships among the supra-sectional clades, and the exact placement of several smaller groups (e.g., Viburnum clemensiae, Viburnum urceolatum, and section Pseudotinus). In two lineages each GBSSI paralogue is represented by two distinct sequences. The presence of additional copies appears to be correlated with polyploidy in these clades. Placement of the homoeologues in our gene trees suggests the possibility of a hybrid origin for these polyploids.

Campylobacter and Salmonella are the most commonly reported pathogens causing foodborne illness in the United States. In turkeys, the potential that semen used for artificial insemination is contaminated with these foodborne pathogens has not been investigated. Because semen on turkey farms is pooled and then used to inseminate multiple hens, contaminated semen could easily spread these bacteria throughout entire flocks via artificial insemination. The objectives of this study were to 1) determine if semen from commercial turkey farms contained these foodborne pathogens and 2) if present, evaluate the efficacy of semen extenders to reduce or eliminate Campylobacter and Salmonella from semen. Semen was collected from randomized pools of ejaculates from 10 to 30 toms per farm from 6 flocks over a 7-wk period and, on occasion, was found to contain Campylobacter, Salmonella, or both. To evaluate the efficacy of semen extenders to reduce or eliminate pathogens, pooled ejaculates were challenged with Campylobacter or Salmonella and treated with commercial poultry extenders containing various concentrations of antibiotics or an antibiotic combination previously demonstrated to remove Campylobacter from mammalian semen. Results demonstrate that commercial turkey semen may contain Campylobacter or Salmonella, and the semen extenders tested either did not reduce the bacteria or reduced but did not eliminate these bacteria from semen. We concluded that semen may be a potential vehicle for Campylobacter transfer to hens, and, if this is true, development of a method for eliminating pathogens in semen before insemination could reduce the risk of colonization.

Campylobacter is one of the most commonly reported bacterial causes of human foodborne infections in the United States. Recent evidence has demonstrated that Campylobacter is present in poultry semen and may contribute to the vertical transmission between the breeder hen and offspring. As Campylobacter is considered sensitive to oxygen and cold temperature, the objective of this study was to determine if aeration and storage temperature could reduce or eliminate Campylobacter in poultry semen. In 4 separate trials, pooled semen samples were collected from roosters or toms, diluted with a commercial poultry semen extender, and inoculated with an average of 10(7) cells/mL of a wild-type C. jejuni or C. coli semen isolate. Pooled ejaculates were then divided into 3 aeration treatments: Control (no aeration), air, or oxygen (gently bubbled for 20 min with atmospheric air or oxygen, respectively). Immediately after aeration, pooled semen samples were further divided to 3 test storage temperatures: 4, 23, or 42 degrees C. At 0, 2, 6, and 24 h of storage, samples were enumerated for Campylobacter. Aeration of the semen alone, or aeration with reduced temperatures (4 or 23 degrees C), did not reduce the amount of Campylobacter compared with the controls. Campylobacter concentrations were, however, reduced when stored at 42 degrees C for 24 h. This effect is associated with reduced sperm viability and is therefore not a practical treatment of reducing Campylobacter in semen. It appears alternative methods will be needed to eliminate Campylobacter from poultry semen.

Summary Background : Silicone percutaneous endoscopic gastros-tomy (PEG) tubes are associated with more short-term complications and possibly fail sooner than those made of polyurethane. Understanding the deterioration and long-term complications leading to tube failure has important clinical and cost implications. Aim : To compare dwell time and patterns of failure ofsilicone polymer and polyurethane PEG tubes. Methods : Participants randomized to receive a siliconepolymer (n = 76) or polyurethane (n = 62) PEG werevisited monthly for 540 days, until death or tube removal. Tube and exit site characteristics and reasons for removal were recorded. Results : Tube failure occurred in 25% of silicone PEGs and 12.9% of polyurethane PEGs. Mean tube survival for silicone PEGs was 377.2 days (CI: 326–428) and for polyurethane 436.8 days (CI: 383–490; P = 0.13). A greater proportion of silicone PEGs developed intralumenal colonization and altered tube integrity than polyurethane (P

Regulatory monitoring for most antibiotic residues in edible poultry tissues is often accomplished with accurate, although expensive and technically demanding, chemical analytical techniques. The purpose of this study is to determine if a simple, inexpensive bioassay could detect fluoroquinolone (FQ) residues in chicken muscle above the FDA established tolerance (300 ppb) comparable to a liquid chromatography-fluorescencemass spectrometryn method. To produce incurred enrofloxacin (ENRO) tissues (where ENRO is incorporated into complex tissue matrices) for the method comparison, 40-d-old broilers (mixed sex) were orally dosed through drinking water for 3 d at the FDA-approved dose of ENRO (50 ppm). At the end of each day of the 3-d dosing period and for 3 d postdosing, birds were sacrificed and breast and thigh muscle collected and analyzed. Both methods were able to detect ENRO at and below the tolerance level in the muscle, with limits of detection of 26 ppb (bioassay), 0.1 ppb for ENRO, and 0.5 ppb for the ENRO metabolite, ciprofloxacin (liquid chromatography-fluorescence-mass spectrometryn). All samples that had violative levels of antibiotic were detected by the bioassay. These results support the use of this bioassay as a screening method for examining large numbers of samples for regulatory monitoring. Positive samples should then be examined by a more extensive method, such as liquid chromatography-fluorescence-mass spectrometryn, to provide confirmation of the analyte.

Better understanding of the functional biology of early angiosperms may clarify eco- logical factors surrounding their origin and early radiation. Phylogenetic studies identify Ambor- ella, Nymphaeales (water lilies), Austrobaileyales, and Chloranthaceae as extant lineages that branched before the radiation of core angiosperms. Among living plants, these lineages may rep- resent the best models for the ecology and physiology of early angiosperms. Here we combine phylogenetic reconstruction with new data on the morphology and ecophysiology of these plants to infer early angiosperm function. With few exceptions, Amborella, Austrobaileyales, and Chlor- anthaceae share ecophysiological traits associated with shady, disturbed, and wet habitats. These features include low and easily light-saturated photosynthetic rates, leaf anatomy related to the capture of understory light, small seed size, and clonal reproduction. Some Chloranthaceae, how- ever, possess higher photosynthetic capacities and seedlings that recruit in canopy gaps and other sunny, disturbed habitats, which may have allowed Cretaceous Chloranthaceae to expand into more diverse environments. In contrast, water lilies possess ecophysiological features linked to aquatic, sunny habitats, such as absence of a vascular cambium, ventilating stems and roots, and floating leaves tuned for high photosynthetic rates in full sun. Nymphaeales may represent an early radiation into such aquatic environments. We hypothesize that the earliest angiosperms were woody plants that grew in dimly lit, disturbed forest understory habitats and/or shady streamside settings. This ecology may have restricted the diversity of pre-Aptian angiosperms and living basal lineages. The vegetative flexibility that evolved in the understory, however, may have been a key factor in their diversification in other habitats. Our inferences based on living plants are consistent with many aspects of the Early Cretaceous fossil record and can be tested with further study of the anatomy, chemistry, and sedimentological context of Early Cretaceous angiosperm fossils.

Comparative embryology is integral to uncovering the pattern and process of metazoan phylogeny, but it relies on the assumption that life histories of living taxa are representative of their antecedents. Fossil embryos provide a crucial test of this assumption and, potentially, insight into the evolution of development, but because discoveries so far lack phylogenetic constraint, their significance is moot. Here we describe a collection of embryos from the Middle and Late Cambrian period (500 million years ago) of Hunan, south China, that preserves stages of development from cleavage to the pre-hatching embryo of a direct-developing animal comparable to living Scalidophora (phyla Priapulida, Kinorhyncha, Loricifera). The latest-stage embryos show affinity to the Lower Cambrian embryo Markuelia, whose life-history strategy contrasts both with the primitive condition inferred for metazoan phyla and with many proposed hypotheses of affinity, all of which prescribe indirect development. Phylogenetic tests based on these embryological data suggest a stem Scalidophora affinity. These discoveries corroborate, rather than contradict, the predictions of comparative embryology, providing direct historical support for the view that the life-history strategies of living taxa are representative of their stem lineages.

Phylogenetic analyses of chloroplast trnK intron and nuclear ribosomal ITS DNA sequences yield signie cant improvements in our understanding of relationships, character evolution, and biogeography in Viburnum (Adoxaceae). We cone rm that most of the ten traditionally recognized sections are monophyletic. The most striking exception is Odontotinus , which is divided into: (i) a purple-fruited New World clade within which the Latin American section Oreinotinus is nested, and (ii) an Old World, mostly red-fruited clade that is closely related to V. cylindricum (section Megalotinus ) and the New World, purple-fruited V. acerifolium . We identify three major supra-sectional groupings: (i) a clade consisting of the Odonto- tinus-Oreinotinus -Megalotinus complex,the circum-boreal section Opulus, andtheEurasiansection Tinus,(ii)acladecontaining the Old World section Viburnum, the New World section Lentago, and, with less support, section Pseudotinus , and (iii) aclade containing the Asian sections Tomentosa and Solenotinus . Two species are not clearly allied to any of these supra-sectional clades: V. urceolatum , a Taiwanese/Japanese species traditionally placed in section Viburnum, and V. clemensiae , a Bornean species previously assigned to section Solenotinus . The placement of the root also remains uncertain, but probably does not fall within any of the three major supra-sectional clades. Knowledge of relationships within sections is useful in clarifying historical biogeography and the evolution of sterile e owers and fruit color. The one case of cone ict between datasets high- lights a possible instance of homoploid hybrid speciation.

Gooseberries are often distinguished from currants as a distinct genus (Grossularia) or subgenus (Ribes subg. Grossularia), but recent molecular phylogenetic analyses of chloroplast and nuclear data disagree as to the monophyly of this group. We report new sequence data from the 18–26S nuclear rDNA ITS and ETS regions and from the chloroplast psbA-trnH intergenic spacer that, in combination with previously reported data, suggest subg. Grossularia is monophyletic and nested within Ribes. Two main lineages are evident within subg. Grossularia, corresponding to the true gooseberries (subg. Grossularia sect. Grossularia) and a clade of glabrous-styled western North American gooseberries (subg. Grossularia sect. Robsonia, subg. Hesperia, Lobbia). Biogeographic analyses based on DIVA optimizations suggest a western North American origin for subg. Grossularia, with subsequent dispersal to east Asia giving rise to a well-supported clade of Asian gooseberry species in sect. Grossularia. This example contr...

The intentional early colonization of the intestinal tract with beneficial microflora, known as competitive exclusion, has been shown to successfully protect poultry from selected enteric pathogens. Although effective cultures have been produced and are available, an inexpensive, air-tolerant, and completely defined culture is needed. Presently, we developed an in vitro competition assay to select for individual facultative anaerobes of poultry enteric origin that could exclude Salmonella. Using this assay, 24 isolates were selected and stored individually. These 24 isolates were amplified in batch culture (tryptic soy broth, 4 h at 40 degrees C) and administered at final dilutions of 10, 100, or 1,000 cfu to day-of-hatch poults. Forty-eight hours later, poults were challenged with 100 to 1,000 cfu antibiotic-resistance-marked Salmonella enteritidis PT 13A by oral gavage. Five days later, all poults were killed, and cecal tonsils were aseptically removed for tetrathionate enrichment (24 h at 37 degrees C) followed by selective plating with marker antibiotics. Selected lactose-negative, antibiotic-resistant colonies typical of Salmonella were further confirmed by serogrouping. Treatment-related protection ranged from 0 to 100% in three experiments. Greatest protection was related to the lowest concentrations of the protective microflora in each experiment. These data suggest that effective combinations of competitive enteric microflora can be identified by appropriate in vitro selection methods.