Daphnia pulex species complex in particular, but no study has completed a wide-scale survey on the crustacean species that inhabit Churchill's aquatic ecosystems using molecular markers. We have employed DNA barcoding to study the diversity of the Branchiopoda (Crustacea) in a wide variety of freshwater habitats and to determine the likely origins of the Churchill fauna following the last glaciation. The standard animal barcode marker (COI) was sequenced for 327 specimens, and a 3% divergence threshold was used to delineate potential species. We found 42 provisional and valid branchiopod species from this survey alone, including several cryptic lineages, in comparison with the 25 previously recorded from previous ecological works. Using published sequence data, we explored the phylogeographic affinities of Churchill's branchiopods, finding that the Churchill fauna apparently originated from all directions from multiple glacial refugia . Overall, these microcrustaceans are very diverse in Churchill and contain multiple species complexes. The present study introduces among the first sequences for some understudied genera, for which further work is required to delineate species boundaries and develop a more complete understanding of branchiopod diversity over a larger spatial scale.The region of Churchill, Manitoba, contains a wide variety of habitats representative of both the boreal forest and arctic tundra and has been used as a model site for biodiversity studies for nearly seven decades within Canada. Much previous work has been done in Churchill to study the  The sub-Arctic region of Churchill, Manitoba, Canada  has been a model ecosystem for numerous biodiversity studies for nearly 70 years Daphnia is the best-studied group in Churchill; attempts to characterize the boundaries and members of the Daphnia pulex complex have evolved from using morphology, to allozyme comparison, to DNA sequencing. Weider and Hebert D. pulex within the Churchill rock bluffs and tundra ponds. Later work by Colbourne et al. D. pulex complex is divided into nine distinct lineages (most of which likely correspond to species) in the Holarctic. Of these nine, only the D. tenebrosa lineage was represented among the individuals they sequenced from Churchill. Weider et al. D. pulex complex alone are found in Churchill, including “panarctic D. pulex”, “western D. pulicaria”, “polar D. pulicaria”, and “D. middendorffiana”. These studies show that without genetic information the understanding of microcrustacean species diversity—and consequently their community associations—is problematic.The majority of genetic studies that shed light upon species boundaries in the Churchill crustacean fauna have focused on the Cladocera, and, more specifically, the daphniids. The genus Sida crystallina consists of four distinct allopatric mitochondrial lineages within North America Simocephalus species Polyphemus pediculusHolpedium gibberum in fact consists of two species: H. gibberum and H. glacialis. Although morphologically similar, these species possess important biological differences. While H. glacialis reproduces in the manner typical for cladocerans , H. gibberum within North America reproduces unisexually, either via selfing or obligate automictic parthenogenesis Holopedium species (or both) inhabits this region, thus limiting our knowledge of community composition, biogeography, and the distributional limits of cyclic parthenogenesis in this genus.While many studies have used allozyme analysis and mtDNA sequencing to characterize individual species or species complexes of other branchiopod groups in North America, little research has been done to analyze the true diversity of the Branchiopoda in the Churchill region. For example, the water flea Daphnia pulex, see D. pulicariac oxidase subunit I (COI) for most animal groups, frequently reveals cryptic species diversity, for example within lepidopterans c. 1180 currently described species of branchiopod crustacean The use of DNA sequences for studying species diversity is now a common practice and is particularly useful for taxa such as the microcrustaceans in which cryptic or nearly cryptic species diversity is very common. Occasionally, deeply divergent lineages that are not even sister taxa are contained within the same species name  for conducting research in the Churchill Wildlife Management Area. The permit numbers are as follows: April 1 2008–March 31 2009 WB08445; April 1 2009–March 31 2010 WB09762. Prior to 2008, the same permits were issued to the CNSC but had no permit number associated with them.Sampling took place in July through the years of 2006–2009 and August 2006 from all types of inland aquatic habitats, including the Churchill River, various creeks, three large lakes, a wide variety of large and small tundra ponds, as well as the rock bluff pools near the shores of Hudson Bay. Collecting methods included both horizontal and vertical plankton tows using a 50 or 250 µm mesh plankton net, as well as free-hand sampling with dip nets from small ponds and littoral zones. Efforts were made to sample both benthic and pelagic species in areas with and without macrophytes present. Specimens were sorted to family, genus, or species when possible and were preserved in 95% ethanol which was changed once after 24 hours. Family and genus-level identifications were performed based on morphology Polyphemus complex Chydorus complex Sida crystallinaDaphnia). We aimed to sequence a sample size of at least 5 specimens per species when possible.Genetic clusters differing from one another by >3% COI divergence were considered provisional species for this study, as that level of divergence is often indicative of different species Specimens were each photographed prior to DNA extraction. Typically, genomic DNA was extracted from entire specimens, but large specimens (>5 mm) were subsampled, with a piece of carapace or one or several limbs used. DNA was extracted and purified following the protocol of Ivanova et al. 2O, 1.25 µl 10× Buffer for Platinum Taq, 0.625 µl of 50 mM MgCl2, 0.125 µl of each primer, 0.0625 µl 10 mM dNTPs, and 0.06 µl Platinum Taq polymerase. Each well contained 2 µl of DNA template. The thermocycling profile consisted of the COIfast reaction, which cycled each plate for one cycle of 1 min at 94°C; 5 cycles of 94°C for 1 min, 45°C for 40 sec, and 72° for 1 min; followed by 35 cycles of 94°C for 1 min, 51°C for 40 sec, and 72°C for 1 min; and then a final cycle of 72°C for 5 min. PCR products were electrophoresed in a 2.0% agarose E-gel (Invitrogen) stained with ethidium bromide and were visualized under UV light.PCR mixes had a total volume of 12.5 µl and contained 6.25 µl 10% trehalose, 2 µl ddHEach PCR product was sequenced bi-directionally on an ABI 3730XL DNA Analyzer (Applied Biosystems). A single consensus sequence was assembled using the forward and reverse sequences using CodonCode Aligner v. 3.0.2 (CodonCode Corporation).A checklist of species previously encountered at Churchill was compiled from the literature , divergences of >5% are typically indicative of different species The distributions of the species or genetic clusters present in Churchill were investigated using a graphical approach using original and published COI data (available from GenBank or BOLD). Polygons were plotted on a map of North America showing the most distant points at which a particular genetic cluster (genetic divergences <2%) has been found to date. We used partially transparent polygons such that areas of overlap are darker; thus, the degree of shading on the map is proportional to the number of lineages likely colonizing Churchill from that region. This exploration of origins makes the assumption that most lineages will display continued occupancy of refugial regions combined with more recent expansion into glaciated regions, rather than large-scale shifts of entire ranges across major regions of the continent.Holopedium sp. we were unable to obtain sequences for these specimens, and so they are not included in any genetic analyses. The overall sequencing success rate was 70% (327/466 specimens), but the high failure rate was mainly due to problematic genera including Lynceus, Holopedium, Bosmina, and Scapholeberis. Of the 139 specimens from which we were not able to obtain sequences at least 350 bp long, 72 of these belonged to these 4 genera alone. Multiple PCR attempts combined with using different primers resulted in success for 3 of these 4 genera. Of the 327 specimens for which we were able to obtain sequences, 294 of them were >500 bp had <1% undetermined nucleotides (Ns), and had two successful trace files, and were thus of barcode quality.In total, 42 species were collected, of which 41 were successfully sequenced  was well represented at Churchill with 15 species encountered , Alona (2), Pleuroxus (2), Picripleuroxus (2), and Acroperus (2), of which the latter 3 genera have not been reported from Churchill previously. The genus Alonella was represented by a single species among our samples.The Chydoridae (Anomopoda) had the greatest diversity with 16 species, 5 of which were in the genus Polyphemus (order Onychopoda) contained 2 species, and the order Ctenopoda was represented only by Sida (1 species) and Holopedium (no sequences obtained). We collected three large branchiopod species, two anostracans  and one laevicaudatan (Lynceus sp.). A notostracan (Lepidurus arcticus) has been previously recorded from Churchill, but we did not encounter this species.Several other groups were present at much lower diversity. The families Bosminidae and Moinidae (Anomopoda) each contained just a single species, while the family Macrothricidae was represented by 2 species. The genus Chydorus and Simocephalus had been reported from Churchill  . Despite these low divergences, all branchiopod species studied displayed no overlap between their own intraspecific and interspecific divergences After cutting the primer sites, the final sequences were 658 bp for those specimens successfully sequenced using Folmer primers. No indels or stop codons were observed in the alignment. The mean intraspecific divergence was 0.52%, while the maximum intraspecific divergence was 3.4% (range 0–3.4%). In most cases maximum intraspecific divergence was <2.4%, with the sole exception being found in 7–32.3%) . The meaPolyphemus pediculus consisted of two clusters separated by a divergence of 15.2%. These corresponded to lineages NA2 and NA3 investigated by Xu et al. Chydorus sphaericus consisted of 2 distinct clusters that corresponded to lineages A3 and B5 found by Belyaeva and Taylor Chydorus brevilabris, C. cf. linguilabris, and Chydorus sp. 2 NA) belonging to the C. sphaericus complex were also found in Churchill. In many additional cases, our results clearly matched the species found in Churchill with lineages investigated by other studies in North America that employed COI. For example, Sida crystallina lineage BER1 Several morphospecies previously recorded from Churchill were found to have deeply divergent genetic clusters, reflecting the presence of cryptic diversity. For example, Daphnia tenebrosa was unique as it was represented by 3 distinct intraspecific clusters separated by averages of 2.5–3.1% divergence   .Simocephalus cf. serrulatus sp.1 NA, Moina macrocopa, Ceriodaphnia cf. laticaudata, Chydorus brevilabris, and Pleuroxus cf. varidentatus), reflecting a southern origin. Other species originated from Beringia, including two members of the Chydorus sphaericus complex (C. sphaericus sp. A3 and sp. B5) as well as the Beringian Sida crystallina lineage. It is not clear whether Daphnia pulicaria and D. cf. middendorffiana colonized Churchill from refugia in the high arctic, or whether they colonized the high arctic from the south after the Pleistocene. The two divergent lineages of Polymphemus pediculus appear to have colonized Churchill from two separate regions, western and eastern temperate North America. Similarly, the two divergent lineages of Simocephalus cf. serrulatus appear to have colonized Churchill from two different regions, northwestern Mexico and eastern temperate North America.Among the 41 species sequenced at Churchill, genetic matches (>98% similarity) were found from other regions for 15 of them. As seen in Daphnia, Chydorus, Polyphemus, Sida), we were successful in matching our results to published sequences, thus creating a more complete understanding of diversity and distributions across a broader area. It appears that post-glacial colonization occurred from both the south and from the west (Beringia), and there may have been some species that arrived in Churchill from high-Arctic North American refugia or from polar Eurasian regions.The purpose of this study was to reveal the species diversity of Branchiopoda from the sub-Arctic region of Churchill, Manitoba, using DNA barcoding as a tool to help delineate provisional species boundaries. Our results show that the fauna in Churchill is much more diverse than previous checklists have shown, with an increase in the total known fauna from 25 to 42 described and provisional species collected during this study alone. We also demonstrated that by using standardized markers, it is possible to study species distributions, biogeographic patterns, and habitat occurrence, which is critical for community ecology studies. In particular, for some better-studied genera  matched to a sequence for a specimen found in the east coastal region, two other species were found on the west coast and the mid-west, suggesting that the southeastern USA may not have been the primary region of Simocephalus diversification in North America. However, there is still a lack of broad geographic coverage of molecular data for Simocephalus.Hann Some branchiopod species show very large distributions in North America. At least 7 of 61 species found in Mexico and Guatemala can be found in Churchill as well, but the majority of species have much narrower distributions In conclusion, this study represents the first intensive DNA barcoding study on all groups of freshwater Branchiopoda from a sub-Arctic site. We found that the region of Churchill, Manitoba, contains higher microcrustacean diversity than previously recorded, and many cryptic, provisional species are likely to be present. While it is difficult to determine what species names should be applied to all genetic clusters, particularly in the absence of comparative data from Eurasia, these gene sequences represent valuable baseline knowledge about this fauna. Some clusters will gain names as comparisons are made, while others likely represent new species; nevertheless, the sequences will remain the same and serve as a record of the species present in Churchill during the timeframe of this study.D. cf. middendorffiana and D. pulicaria. While these results may simply reflect current species distributions, it is likely that most of these represent colonization events from those refugial regions due to the fact that some species (e.g. Chydorus sphaericus sp. A3 and sp. B5) have only been found near specific refugia (in this case Beringia) despite broad study By matching COI sequences of some well-studied species, we were able to infer the phylogeographic origins of some of the Churchill fauna. It appears that Churchill was colonized from multiple directions following the Pleistocene glaciations, including the Beringian refuge, the southeastern and southwestern United States, as well as potentially a high arctic refugial area for Future studies should focus on understudied groups for which there is currently little other molecular data. Many species were only given interim names due to the fact that many cryptic species have not been identified and described formally, and limited comparative data exist for many genera. It should also be a priority to formally describe species and to determine whether our shallower COI clusters represent species or glacial phylogroups using other life history, morphological, and ecological traits. Further genetic work on the Branchiopoda will help uncover their true diversity and distributions on a global scale. In particular, much more work is needed on the small-bodied, primarily benthic groups (such as Chydoridae) as our study revealed substantial diversity in this group. A comprehensive survey of DNA barcodes from different geographic regions will facilitate more objective research on the ecology, community associations, and environmental tolerances of aquatic biotas.Table S1List of species found in Churchill from the literature versus the species revealed by DNA barcoding. The results of our study show a large increase in the number of species compared to the number found in published literature on Churchill branchiopods. The type of habitat where each species was found is also listed. References are listed for both the original Churchill literature and for the studies from which we were able to obtain sequence matches of ≥98%. Daphnia magna and D. tenebrosa were identified morphologically and also had matches of ≥96% to published sequences.(DOCX)Click here for additional data file.Table S2Summary of COI divergence patterns for all species with a sequence length >350 bp. The mean and maximum intraspecific divergence found in all species which were successfully sequenced. The distance to the nearest neighbour is also provided, along with the sample size.(DOCX)Click here for additional data file.


Cynometra alexandri constitutes only 9.6% of all trees in the gallery forest in which the study populations ranges, but it was selected for 73.6% of the 1,844 chimpanzee night beds we sampled. To determine whether physical properties influence nesting site selection, we measured the physical characteristics of seven common tree species at the Toro-Semliki Wildlife Reserve, Uganda. We determined stiffness and bending strength for a sample of 326 branches from the seven most commonly used tree species. We selected test-branches with diameters typically used for nest construction. We measured internode distance, calculated mean leaf surface area (cm2) and assigned a tree architecture category to each of the seven species. C. alexandri fell at the extreme of the sample for all four variables and shared a tree architecture with only one other of the most commonly selected species. C. alexandri was the stiffest and had the greatest bending strength; it had the smallest internode distance and the smallest leaf surface area. C. alexandri and the second most commonly selected species, Cola gigantea, share a ‘Model of Koriba’ tree architecture. We conclude that chimpanzees are aware of the structural properties of C. alexandri branches and choose it because its properties afford chimpanzees sleeping platforms that are firm, stable and resilient.The daily construction of a sleeping platform or “nest” is a universal behavior among large-bodied hominoids. Among chimpanzees, most populations consistently select particular tree species for nesting, yet the principles that guide species preferences are poorly understood. At Semliki,  Juvenile and adult apes devote considerable time and energy to the construction of a new sleeping platform or “nest” at the end of their daily active period sleep quality hypothesis holds that apes construct sleeping platforms to allow uninterrupted sleep and to promote longer individual sleep stages, resulting in a higher sleep quality predation avoidancepostural stability  at three relatively dry sites  as raw material for sleeping platform construction. C. alexandri is common in dry or riverine forests throughout Central and East Africa Branch diameters of supporting structures have been shown to be an essential variable for understanding sleep site selection and canopy movement in orangutans, as local diameter is negatively related to compliance and positively related to length of the trunk s Ishaha  in westeA body mass of 30 kg or more means that chimpanzees require strong materials to contain their center of mass during sleep phases with a period of physical paralysis Authorization to conduct research inside Uganda was granted by the Government of Uganda. Permission to carry out research at the Toro-Semliki Wildlife Reserve was granted and approved through permits from the Uganda Wildlife Authority (UWA) and the National Research Council.2 in the Great Rift Valley from the Semliki River in the west to the top of the escarpment in the east, and from the foothills of the Ruwenzori Mountains in the south to the shore of Lake Albert in the north, northwest of Fort Portal, Uganda . The Wasa River runs from south to north through the center of the reserve, emptying into Lake Albert in the north. The reserve is crosscut by small, shallow, often seasonal water-courses that support narrow gallery forests ranging in width from 50–250 m 2 (minimum convex polygon). The biome is predominantly dry Combretum and Borassus palm grassland with only 7.25% forest cover The Toro-Semliki Wildlife Reserve (TSWR) occupies 548 kmPhenology data on tree species distribution throughout the habitat was used from previous work Model of Attim (9.9%), Model of Fagerlind (5.3%), Model of Koriba (58.2%), Model of Leeuwenberg (10.4%), Model of Tomlinson (4.4%), and Model of Troll (0.4%). We used 2X analysis to assess chimpanzee preference for architectural models, relative to prevalence within the home range.We followed van Wyk and colleagues Beilschmiedia ugandensis was an ideal control given its rare selection as a sleeping site and ubiquity throughout the home range and was featured in a LSD post hoc analysis of the RBF values compared among preferred species.Our methods closely followed Stewart and colleagues Beilschmiedia ugandensis as a control. We calculated the surface area of leaves (N = 428) from selected tree species by tracing leaf outlines over 1 cm2 paper (then summing the number of squares within the outline) and compared species using a one-way ANOVA, again with B. ugandensis used as a control.We measured internode lengths (N = 2574) along the stems of selected tree species often selected for sleeping sites to compare internode distances among genera (One-way ANOVA), using All statistical tests were 2-tailed, set at an alpha = 0.05 significance level.Cynometra alexandri was selected for 73.6% of the nests, even though it represents only 9.6% of all trees on our 20 habitat-sampling transects  greater than the other seven species in three of four diameter categories (see P<0.001), category 2, 3–4.9 cm , category 3, 5–6.9 cm . Size category 4, 7–9 cm, (F P = 0.524) did not differ significantly from other species; however, our sample contained very few branches in this diameter category . In a one-way ANOVA C. alexandri had significantly smaller leaf surface areas than other species . The second most commonly selected tree species, C. gigantea, had the largest leaf surface area .In a one-way ANOVA, Model of Troll ; Model of Leewenberg (both Combretum [N = 24] and Ficus [N = 30]); Model of Tomlinson (Phoenix [N = 70]); Model of Koriba . Koriba was the most preferred tree architecture, selected for 90.0% of all trees, significantly greater than expected by chance  Our results strongly suggest that just as do orangutans C. alexandri had the smallest leaves with the greatest density  of all nesting tree-species, which has been suggested as affording the greatest comfort C. alexandri may also have insect-repellent properties Phoenix reclinata, a palm with physical characteristics dramatically different from the most-preferred species. P. reclinata has pinnate, or feather-shaped leaves 3–5 cm in breadth and 40 cm in length. The leaves are stiff and the base of the leaf stem has thorns that incorporate paralytic secondary compounds Among the preferred tree species at Semliki  was PhoeDrypetes gerrardiiC. alexandri tree architecture affords chimpanzees inverted-tripod nest frames, which are hypothetically the easiest to transform into the functionally concave form to maximize comfort and safety C. alexandri likewise support this hypothesis: three of the four  tree architectures used by chimpanzees at Semliki possess inverted tripod branching patterns (Tomlinson is a unique case, as it is an oil-palm tree with a markedly different shoot development.Our results are compatible with the hypothesis that chimpanzees select trees that are configured similar to the lollipop morphology of patterns . The treC. alexandri has all of these properties, suggesting that chimpanzees are keen observers of physical properties of trees, including stiffness, strength and leaf surface area, and that they select species that provide the widest range of advantages, including predator avoidance, postural stability, thermoregulation and pathogen avoidance.Our results suggest that the ideal sleeping platform tree species might well possess multiple advantages, perhaps possessing antipredator, antivector, thermoregulatory and comfort- maximizing qualities all at once.


Colony formation was the first step towards evolution of multicellularity in many macroscopic organisms. Dictyostelid social amoebas have used this strategy for over 600 Myr to form fruiting structures of increasing complexity. To understand in which order multicellular complexity evolved, we measured 24 phenotypic characters over 99 dictyostelid species. Using phylogenetic comparative methods, we show that the last common ancestor (LCA) of Dictyostelia probably erected small fruiting structures directly from aggregates. It secreted cAMP to coordinate fruiting body morphogenesis, and another compound to mediate aggregation. This phenotype persisted up to the LCAs of three of the four major groups of Dictyostelia. The group 4 LCA co-opted cAMP for aggregation and evolved much larger fruiting structures. However, it lost encystation, the survival strategy of solitary amoebas that is retained by many species in groups 1–3. Large structures, phototropism and a migrating intermediate ‘slug’ stage coevolved as evolutionary novelties within most groups. Overall, dictyostelids show considerable plasticity in the size and shape of multicellular structures, both within and between species. This probably reflects constraints placed by colonial life on developmental control mechanisms, which, depending on local cell density, need to direct from 10 to a million cells into forming a functional fructification. In many and perhaps all cases, colony formation may have been the first step towards multicellularity . AlthougWe investigate molecular changes that allowed colonial organisms to achieve greater levels of multicellular complexity. Dictyostelid social amoebas offer unique opportunities to resolve this problem. They are a genetically diverse group , which cIn this work, we have measured 21 traits that were partially covered by the original diagnoses and we investigated deeper traits, such as the alternative survival strategy of encystation, the ability to form motile ‘slugs’ and the identity of the signals that coordinate cell movement during aggregation and morphogenesis. The dataset of phenotypic traits was submitted to phylogenetic comparative analysis to retrace character history and identify correlated evolution of characters.2.(a)(i)Klebsiella aerogenes [K. aerogenes on one-fifth standard medium or one-third lactose-peptone agar with charcoal for robust and delicate species, respectively .For analysis of morphology, species were cultured on non-nutrient (NN) agar with pregrown erogenes . For oth(ii)in situ from agar plates, using a Leica MZ16 stereo microscope, equipped with graticule. The morphological characteristics of amoebas and stalks were assessed by transferring cells and structures to a droplet of phosphate buffer (PB)  on a slide glass. Spore dimensions were measured from printed images. Each feature was measured in 25–50 individuals of each species.The shape and dimensions of multicellular structures were mostly assessed or measured (iii)7 or 108 cells ml−1 on agar and incubated under unilateral illumination. After 1–8 days, the developing structures were scored for phototaxis or phototropism.Amoebas were deposited as 10 µl streaks, containing 3 × 10(iv)7 or 3 × 107 cells ml−1 in PB and incubated for up to 36 h as 10 µl droplets on NN agar containing Sp-cAMPS , glorin , folate (Sigma) or neopterin  with the solvents of either compound as controls.Amoebas were resuspended at 10(b)(i)coffee [rBayes v. 3.2 [axML in TOPALi [The sequences of 32 orthologous proteins were retrieved from six dictyostelid and three amoebozoan genomes and aligned using M-coffee . The cons v. 3.2  and RaxMn TOPALi . A rooten TOPALi  to the 3(ii)The SSU rDNA_32 protein phylogeny  was combined with the matrices of continuous or coded categorical characters . For continuous traits, ancestral states were estimated by a maximum-likelihood-based method under a Brownian motion model of evolution . All tra(iii)esquite [p-values) of the log likelihood differences were estimated with 100 simulations. To correct for wrongly rejected null hypotheses in multiple comparisons, the threshold p-value for rejecting the null hypothesis (no correlation between datasets) was adjusted by a false discovery rate-based method [Correlated evolution of quantitative characters was determined using phylogenetic generalized least squares , as impesquite . ‘Discred method .3.(a)(i)xcel file ‘Trait_Analysis’, sheet 2. The range of averaged values per species was subdivided into four intervals, such that each interval contained an equal number of species. These intervals, the character states, are plotted onto the previously constructed SSU rDNA phylogeny [Growing amoebas are morphologically similar in all dictyostelids, and show filose pseudopodia, prominent food vacuoles and contractile vacuoles . Spores,hylogeny  in the ea,b). Spores were markedly smaller in group 1 (c) and usually harboured consolidated granules in groups 1 and 3, and unconsolidated granules in clade 2B (l). Most species in clade 2A have round spores, whereas the oblong spores of group 4 species mostly lack prominent polar granules.Amoeba diameters range from 4 to 17 µm between species, but there are no marked differences in amoeba dimensions between groups a,b. Spor group 1 c and usuclade 2B l. Most s(ii)m). Aggregates are on average larger in groups 4 and clade 2B than in groups 1, clade 2A and 3 (e). Aggregates transform into one or more fruiting body precursors, the sorogens or slugs. Slugs are also relatively large in groups 4 and clade 2B, and this trend continues in the dimensions of the stalk and spore head (sorus) of the fruiting body (f–k). Despite these trends, there is large size variation between multicellular structures within species , illustrating that species can accommodate greatly varying amounts of cells within a single structure.Cells can either aggregate as individuals forming a mound, or join up to form inflowing streams, which sometimes fragment into accessory aggregates. Intermediate forms, where cells first aggregate individually and then form streams, or do both, also occur. In group 4, species always aggregate in streams, whereas species in the other groups show a mixture of aggregation types m. Aggreg2A and 3 e. Aggreging body f–k. Despn). Fruiting body habit and branching pattern also show group-specific trends, with group 4 species mainly forming solitary, unbranched fruiting structures, while multiple loosely (gregarious) to tightly (coremiform) grouped fruiting bodies emerge from group 1–3 aggregates . Lateral branching is common in groups 1 and 3, while most clade 2B species and two species that are intermediate to groups 3 and 4 form regular whorls of branches .Sorogens either form fruiting bodies directly or migrate horizontally, often leaving the stalk behind. Migration is mostly absent or weak in groups 1–3 and common in group 4 n. Fruitiq). The stalk base is usually round to conical, but a small clade of group 3 species split the stalk in sections to form a crampon . Several group 4 species form a cellular basal disc or supporter to buttress the stalk (figure 1s).In most Dictyostelia, the stalk consists of highly vacuolated cells with a cellulose wall, except for clade 2A, where the stalk is a cellulose tube . The stalk tip is commonly broadened (clavate or capitate) in groups 1 and 4, pointed (acuminate or piliform) in clades 2A and 2B, and a mixture of those in group 3 q. The st(iii)t; electronic supplementary material, figure S3). One-third of group 1 and half of group 3 species showed sparse to moderate encystation. Remarkably, no group 4 species could be induced to encyst.Similar to their amoebozoan ancestors, several dictyostelids can still encyst individually under conditions that do not favour aggregation . To invefigure 1(iv)u; electronic supplementary material, figure S1). About 25 per cent of group 1 and 40 per cent of group 3 species showed strong phototropism and over 60 per cent of group 4 species.Both the migration of slugs and the outgrowth of fruiting bodies are often oriented towards light. These behaviours, termed phototaxis and phototropism, respectively, direct the structures to the soil surface, where spores are readily dispersed . We analfigure 1n), but was sometimes worse and sometimes better under unilateral light. In group 4, approximately 70 per cent of species migrate strongly and these species are all phototactic. All migrating group 3 species are phototactic and most of the clade 2B species, but in groups 1 and clade 2A, both migration and phototaxis are weak or absent .Phototaxis requires the ability of slugs to migrate. Migration was also scored during development under incident light n, but war absent v,w.(v)Dictyostelium discoideum and some group 4 species use cAMP; the Polysphondylium violaceum chemoattractant is glorin, a modified dipeptide [Dictyostelium minutum and Dictyostelium lacteum chemoattractants were identified as folate and neopterin, respectively [D. discoideum [The chemoattractants for aggregation are only known for a few species. ipeptide , and theectively ,28. Folascoideum . ChemoatAll species were spotted on agar containing increasing concentrations of glorin, folate, neopterin or Sp-cAMPS. The latter was used instead of cAMP to prevent hydrolysis by secreted phosphodiesterases . Developx and 1y and P. violaceum and its two relatives, Dictyostelium laterosorum and Polysphondylium patagonicum, which occupy a small clade between groups 3 and 4, was disrupted by 1 or 10 µM glorin, as was aggregation of several species in groups 1–3. There was no effect of folate, glorin and neopterin on culmination that could not be attributed to an earlier effect on aggregation. Asghar et al. [Sp-cAMPS inhibits aggregation of all group 4 species, but none of the species in groups 1 and 2 (i)Dictyostelium purpureum (Dpur) from group 4 [D. lacteum (Dlac) from group 3 (http://sacgb.fli-leibniz.de/cgi/index.pl) and Acytostelium subglobosum (Asub) from clade 2A (http://acytodb.biol.tsukuba.ac.jp/). Additionally, Amoebozoan genomes became available that provide less distant outgroup sequences, such as the genomes of Entamoeba histolytica [Acanthamoeba castellani [Physarum polycephalumhttp://genome.wustl.edu/genomes/view/physarum_polycephalum. We retrieved orthologues for all or most of the 32 genes of the previous set of 33 genes from the six dictyostelid and three amoebozoan genomes and prepared a concatenated alignment of about 18 180 amino acids, which was subjected to different methods for phylogenetic inference. All methods robustly placed the root between two branches that contain groups 1 and 2, and groups 3 and 4, respectively . This topology was also produced using three out of four concatenated sets of seven of the shorter proteins , but by only two of the four remaining larger proteins , while SSU rDNA yielded the same topology  as previously [E. histolytica sequences diverged strongly from the other amoebozoan sequences, possibly owing to its anaerobic, parasitic lifestyle. Deleting these sequences did not affect the rooting  and they were omitted from subsequent inference of a phylogeny for all Dictyostelia. This phylogeny was inferred from the earlier alignment of the SSU rDNAs of all species [Dictyostelium polycarpum, which was previously located between groups 2 and 3, is now a sister species to group 2. This is probably not artefactual, since D. polycarpum displays morphological features, such as unconsolidated spore granules and pointed stalk tips that are characteristic features of group 2 .A reliable phylogeny is of primary importance to retrace phenotypic evolution. The earlier SSU rDNA and β-tubulin-based phylogenies subdivided dictyostelids into the same grouping, but did not agree about the root of the tree, with rDNA pointing to group 1 and α-tubulin to group 2 as the earliest diverging group . A phylo group 4 , D. lacttolytica , Acanthastellani  and Physeviously . The E.  species ,12, conc species ,12, but (ii)c. The inference shows that the LCA to all Dictyostelia formed relatively small sorogens and fruiting bodies. The LCAs to groups 1–3 retained their small size, but the group 4 LCA increased fruiting body size about 2.5-fold (figure 3c). However, within each group, except for clade 2A, larger forms emerged, as illustrated for sorus diameter in figure 3b.The newly inferred phylogeny was combined with the characters measured in this work to infer the phenotypes of the last common ancestors (LCAs) of major groupings. Ancestral states for continuous traits were reconstructed using a method based on maximum likelihood  and are figure 3a–p). For several labile characters, such as aggregate and stalk base shape , the ancestral states at many major nodes are equivocal, presenting roughly equal likelihoods for all different states. Characters for which ancestral states at major nodes could be inferred with 60–100% probability are summarized in figure 3c. The LCA to all Dictyostelia formed unbranched grouped fruiting bodies , with a cellular stalk and broadened stalk tip . Its spores were elliptical and contained polar granules . It used cAMP to coordinate fruiting body morphogenesis , but not aggregation , which may have been mediated by glorin .Except for the broadened stalk tip, these character states persisted into major branches I and II, and into the LCAs of groups 1–3. The LCA to group 2 gained pointed stalk tips, while the LCA to clade 2A also gained an acellular stalk. The LCA of clade 2B adorned its fruiting bodies with regular whorls of side branches, while the LCA of clade 2A lost polar spore granules. The group 4 LCA also lost polar spore granules, while its sorogens acquired migratory behaviour and its amoebas used cAMP for aggregation. It should be noted that all inference of ancestral states reflects probable trends in trait evolution, which inevitably involves uncertainty, and not a definitive trait history.(iii)To gain initial insight into possible common causes for character evolution and causal relationships between characters, we investigated to what extent changes in individual characters are correlated. Standard statistical correlation methods are not appropriate, because species have varying degrees of shared ancestry and cannot be considered as independent samples –36. To aThe most obvious set of positively correlated traits are the dimensions of aggregates, sorogens and various parts of the fruiting body as the sorus, stalk and stalk base . This is not surprising since large aggregates will normally give rise to large fruiting structures, unless they split up to form multiple sorogens. There is a weak positive correlation between the size of spores and the size of amoebas, and between the size of either amoebas or spores and the size of multicellular structures. The variance in amoeba size explains 12 per cent of the variance in spore size and 4–13% of the size variance in multicellular structures. This suggests that amoeba size contributes to the size of spores and structures, but is not the major determinant.The categorical characters form a much larger dataset and to correlate these characters with each other and with the continuous characters, we used Pagel's ‘Discrete’ test , which cThe strong positive correlations between the dimensions of multicellular structures that were detected with PGLS are also found with ‘Discrete’, indicating that the reduction from continuous to binary character states has no profound effect on the outcome of the analysis. Large size of structures is negatively correlated with a clustered habit, consolidated spore granules and encystation. Mostly, size is not or negatively correlated with dominant branching. However, stalk length is positively correlated with the formation of regular whorls of branches. Interestingly, the size of structures is positively correlated with cAMP-mediated streaming aggregation and with the cellular stalk, slug migration, phototaxis and phototropism, with the latter two features very strongly correlated with each other.4.(a)Polysphondylium pallidum, can form fruiting bodies from as few as seven cells [The traitmap of all measured characters  shows a fairly scattered distribution of character states over the tree with many states reappearing multiple times in different clades. This is particularly the case for features that describe the size and shape of aggregates, slugs and fruiting bodies. Most species are also individually polymorphic for these characters . Depending on local conditions of cell density, individual species can make aggregates and fruiting bodies in a wide range of sizes. Even when developed under standardized conditions, individual structures from the same species easily show a fourfold difference in size . At low food availability, even a fairly large species, such as en cells .The morphology of structures also varies depending on cell density. Species, which normally have clustered or branched fruiting bodies show solitary and unbranched phenotypes when developing from small numbers of cells, while normally solitary and unbranched species show some clustering and branching at high cell density.D. discoideum, and representative taxa from all four groups is coordinated by cAMP pulses that are emitted by the tips of sorogens and propagate through the structure as standing or spiral waves [There is good evidence that fruiting body morphogenesis in al waves ,37–40. Tal waves ,42. The al waves –45. Such(b)Despite morphological plasticity, there are trends in dictyostelid phenotypic evolution. Ancestral state reconstruction showed that the LCA to all Dictyostelia as well as the LCAs of the two major branches and groups 1–3 probably had small, unbranched fruiting structures, containing elliptical spores with polar granules. cAMP was probably used to coordinate fruiting body morphogenesis, but not aggregation, which could have been mediated by glorin (et al. [Correlation analysis highlights coevolution of large, solitary, unbranched multicellular structures, streaming aggregation, slug migration, phototaxis and phototropism, and use of cAMP as attractant . The anaet al.  proposedet al. . Lack ofA more enigmatic relationship that was already noted in the 1970s  is the cThe future aim of our work is to identify causal relationships between the evolution of multicellularity and the evolution of genes and genomes. Bioinformatic and experimental approaches are being used to analyse changes in content, regulation and function of developmental control genes between the recently sequenced genomes of species representing all major groups of Dictyostelia. Combined with the opportunity to replace genes in both late and early diverging species with more ancestral or derived alleles, respectively, such approaches have already yielded insight into the evolutionary history of cAMP signalling  and can


Pseudomonas aeruginosa, and house dust account for 88.0% to 100% of the observed positive reactions to the 62 types of allergenic sources tested. The  Kolmogorov-Smironov Test results of the website-obtained allergen data and allergen family featured peptides suggested that allergen research in laboratories worldwide has been conducted in parallel on many of the same species. The major allergens were reduced to 21 representative allergens, which were further divided into seven structural classes, each of which contains similar structural components. This study therefore has condensed numerous allergenic sources and major allergens into fewer major representative ones, thus allowing for the use of a smaller number of allergens when conducting comprehensive allergen testing and immunotherapy treatments.Vast amounts of allergen sequence data have been accumulated, thus complicating the identification of specific allergenic proteins when performing diagnostic allergy tests and immunotherapy. This study aims to rank the importance/potency of the allergens so as to logically reduce the number of allergens and/or allergenic sources. Meta-analysis of 62 allergenic sources used for intradermal testing on 3,335 allergic patients demonstrated that in southern China, mite, sesame, spiny amaranth,  Over the years, vast amounts of data regarding worldwide allergy diagnosis and treatment have been accumulated. More recently, allergen researchers have probed this data resulting in the complete cDNA sequencing of a large number of allergens, as well as the determination of their three-dimensional 3D) structure in some cases. However, systematic evaluation of the importance and/or potency of the allergens or their sources has not been investigated thus complicating the identification of specific allergenic proteins when performing diagnostic allergy tests. On the other hand, many patients react to a large number of proteins, and allergic cross-reactivity has been described on many levels . The relD structuOur previous results showed that 478 allergens retrieved online could be clustered into eight groups, regardless of their biological source . It woulIn the present study, we reviewed our clinical intradermal test (IDT) data from 2001 to 2003 on 62 kinds of allergenic sources and compared the amino acid sequences and 3D structures of the major allergens obtained from different biological sources, with the goal of logically reducing these allergenic sources to a few species, as well as progressively clustering the 280 major allergens obtained from the ExPASy Proteomics Server  into several of the most representative major allergens. The results obtained could therefore facilitate more consistent and straightforward allergen research.Over the past 32 years, we have intradermally tested (IDT) more than 90,000 patients referred to our allergy clinic. The IDT results of 62 allergen extracts on 3,335 patients in a three-year period (2001–2003) are shown in All allergens extracts were prepared in a sterile environment followed by toxicity and potency evaluation according to an in-house standard protocol as described . After fμL of each allergen dilution was intradermally injected into each patient with 24–30 allergens tested at a time. Injected allergens were arranged vertically on the upper arm(s) with an interval of 2.5–3.0 cm. A positive control  and a negative control (solvent) were also included, and there was no duplicate testing. After 15 min, the size of the wheal was determined by measuring the diameter in two perpendicular directions and then halving the sum. Since bacterial proteins always exhibit a late phase reaction, those results were measured after 24 hrs. The reaction was regarded as positive if the calculated wheal diameter was more than 5 mm.On the day of the test, the allergen extracts were brought to room temperature and diluted immediately in the solvent . Each patient received IDT using the same batch of allergen extracts. IDT was performed as follows: 10–20 To assess whether the distribution of positive reactions to each allergenic source reflects a correlation between different allergenic sources, the number of patients with positive IDT reactions to each allergenic source was counted. Patients showing multiple positive reactions to different allergenic sources were counted once for each individual allergenic source they reacted to. All the allergenic sources were ranked according to patient counts, since there may be cosensitization to different allergenic sources.http://www.allergen.org/. The overall research status of allergens worldwide was analyzed as follows: the number of allergenic species and the related allergen numbers within each taxonomic category were recorded. The consistency of the two distributions thereof was investigated by Kolmogorov-Smirnov test, which measures the maximum difference between two cumulative distribution functions and calculates the probability that the two observed distributions would exhibit a difference at least that large if the samples were drawn from identical populations [Dn,n1~n2 = Maximum  (Sn1 | x | −Sn2 | x|), n = n1∗n2/(n1 + n2), where n1 and n2 denote the number of allergenic species and the number of allergenic proteins within each taxonomic category, respectively.The nonredundant allergen data were extracted from the IUIS allergen list  available at the website ulations . The parhttp://www.expasy.org/) using the keyword “major allergen” and was selected from the IUIS allergen list  by IgE-binding potency. The phylogenetic relationship among the major allergens was inferred by the free package Clustal W 1.83 [Another set of data related to the amino acid sequences of major allergens was retrieved on June 20, 2012, by searching UniProtKB/Swiss-Prot  are located. AFFPs are allergen-specific peptides panned from nonredundant allergens and harbor perfect information with noise fragments eliminated because of their similarity with nonallergens. 534 AFFPs can correctly discriminate 2290 allergens at the highest sensitivity and specificity and make the underlying software SORTALLER outperform other methods at present , which dTo compare the three-dimensional (3D) structures of the major representative allergens, the 3D structure of each major representative allergen was modeled in SWISS-MODEL workspace, a web-based integrated service dedicated to protein structure homology modeling and assessment that can be accessible via the ExPASy Bioinformatics Resource Portal . The modDermatophagoides pteronyssinus and D. farinae) were the number one allergenic source, resulting in more than 70% of the positive reactions among all the four groups. Reaction to sesame (Sesamum indicum L.) ranked second with 49.3% of the positive reactions. Spiny amaranth (Amaranthus spinosus L.), Pseudomonas aeruginosa, and house dust were next with 49.2%, 48.3%, and 44.2% of the positive reactions, respectively. In total, these five allergenic sources express 88.0% to 100% of the positive reactions when compared with microbial allergens, 90.6% to 100% when compared with food allergens, 96.3% to 100% when compared with contact allergens, and 99% to 100% when compared with pollen allergens. That is to say, these five allergenic sources are the most potent and show the most reactivity among the patients we tested; hence we have designated them as the major representative allergenic sources.Intradermal tests using 62 allergenic sources were performed on 3,335 allergic patients. 3,084 of these patients reacted to at least one of the allergenic sources, with the positive frequency being 89.8%. All the allergenic sources used were aligned according to the number of patients with positive reactions. For convenience, the 62 allergenic sources were divided into four general groups, that is, contact allergens, food allergens, pollen allergens, and microbial allergens. The top seven most potent allergenic sources across all groups were then analyzed as to the frequency of their positive reactions when compared to the positive reactions seen with the specific allergens in any particular group. As shown in http://www.allergen.org/), relating nine categories and 241 species altogether  between the number of allergenic species in each category and the number of allergens in each category , 727 nonredundant allergens were listed in the allergen website  were represented by single entries. This method allowed us to reduce 534 AFFPs into 21 allergens  through  Malassezia furfur [3D structure modeling was completed for most of the above 21 allergens by an automated mode or template identification mode in a SWISS-MODEL workspace . Only O8a furfur , had no a furfur  and O058β-barrel: includes P80384, the major allergen Lep d 2 from Fodder mite (Lepidoglyphus destructor) [ Dermatophagoides farinae [Up-and-down tructor) , as well farinae .β-meander and/or ψ-loop constituted calyx, shortened as β(α) calyx, such as hexagon, cradle, and globose twins: includes allergens Q01940 [s Q01940 , P18632 s Q01940 , Q40967 s Q01940 , Q84 UI0s Q01940 , and O82s Q01940 .α-β structured crane: includes Q8I9R5, an allergen from Sarcoptes scabiei type hominis [ hominis .α-β arranged banana string: includes Q95182 [s Q95182  and P431s Q95182 .α(β) formed complex: includes P08176 [s P08176  and Q068s P08176 .α-helix built clips: a large group including P30438, P30440, P59747 [, P59747 , Q40237 , P59747 , O04404 , P59747 , Q9M5X7,, P59747 . Interesα-helix spiral cord: includes P01501 [Apis mellifera), also a main toxin of bee venom with strong hemolytic activity. Q95WY0 is the major oyster allergen and tropomyosin from the pacific oyster Crassostrea gigas. These two allergens exhibited similar structures but displayed different lengths of their spiral cord.s P01501  and Q95Ws P01501 , both ofWhen inspected from spatial structural orientations and surface exposures of the allergens, all of the 21 allergens were shuffled against the initial clustering and interestingly fell into seven structural classes . HoweverThere is a tight link between allergen diagnosis and immunotherapy. After obtaining the results of allergen intradermal testing, specific IgE diagnosis, and/or even challenge assays, those allergen(s) with the highest positive scores would typically be chosen for use in immunotherapy on the allergic patients. The advent of molecular biology and bioinformatics heralded an unprecedented breakthrough in the development of recombinant allergens engineered to have the same immunological characteristics as natural allergens. Many studies have therefore used recombinant allergens in place of their natural counterparts. Unfortunately, allergen screening from one species to another in parallel only displays an ostensible prosperity of allergen study and could not pinpoint the importance of each allergen. The accumulation of a large quantity of overlapping data is threatening to undermine the achievements of allergen research. Whereto allergen research goes becomes a compelling question.Sesamum indicum L.), spiny amaranth (Amaranthus spinosus L.), Pseudomonas aeruginosa, and house dust are the five most prevalent allergenic sources and can well represent the 62 allergenic sources identified in southern China.Facing this question, we firstly cast a meta-analysis on 62 allergenic sources used for intradermal testing on 3,335 patients in a three-year period. The result demonstrated that 88% to 100% of the patients were cosensitized to the top five allergenic sources and assumed that these five allergenic sources would have positive immunotherapeutic effects on the majority of the patients and that the remaining allergenic sources would have minor effects on the patients when used for immunotherapy. All these data corroborate that mite, sesame  and Poaolcalcin ), cerealolcalcin ), nonspeolcalcin ), and evolcalcin ) and Oleolcalcin ). Major olcalcin . All theolcalcin , 47 and The 3D structures of a continuously increasing number of allergens are currently being solved. Therefore, it is possible to produce recombinant allergens that exactly mimic their natural wild types and even to produce genetically engineered hypoallergens with nil or low IgE reactivity but retained T-cell reactivity. As the present study demonstrates, clinical cosensitization to multiple allergenic sources can be attributed to a few major allergenic sources; major allergens from different species can be logically reduced to 21 major representative allergens and even grouped into 7 or less structure classes with similar structure scenarios shared by different allergens. It is tempting to think that there might not be a need to unequivocally search all the undeveloped species for totally novel allergen genes or to equivalently test numerous allergenic sources on patients. Thus, it is cost-efficient, practical, and crucial to have the recombinant allergen research focused on the major representative allergens or core AFFPs for immunotherapy strategy development and diagnosis formulation.We recently noticed that single or fewer major allergens can not only be used to diagnose the genuine sensitization of patients to a given allergen or to the cross-sensitization to several allergenic sources, but also be used for allergen-specific immunotherapy to yield the same effects as the whole allergen mixtures in allergic patients , 48, 49.


Aythya ferina), a long-distance migratory duck and potential vector of highly pathogenic avian influenza. We collected several hundred samples from breeding and wintering grounds across Eurasia including some H5N1-positive individuals and generated partial sequences of the mitochondrial control region and multilocus microsatellite genotypes. Genetic differentiation among breeding populations was significant for both marker types but higher for maternally inherited mtDNA than for biparentally inherited nuclear markers. There was only weak genetic divergence between ducks sampled in Europe and East Asia, and genetic differentiation between populations was not generally associated with geographical distance. No evidence of genetic substructure was detected for ducks sampled on the European wintering grounds. Our results suggest limited breeding-site fidelity, especially in females, but extensive population admixture on the wintering grounds. The specific role of pochards as natural vectors of zoonotic pathogens and in particular H5N1 remains to be clarified but our results point to wintering grounds as potential hotspots for disease transmission.An understanding of the distribution and spatial structure of the natural vectors of zoonothic pathogens is of interest for effective disease control and prevention. Here, we investigate the range-wide population genetic structure of common pochard ( Zoonoses are diseases that are transmitted from animals to humans . Most zoAnas acuta) wintering in Japan and North America, respectively, share breeding areas in eastern Russia (Odocoileus virginianus), a vector of chronic wasting disease  dynamics of associated pathogens e.g., . In partn Russia . This spn Russia . At a mo disease . Consequ disease .Aythya ferina, pochards hereafter) have been listed as a high-risk species in relation to AI by the European Union (2005) because they were repeatedly detected as carriers of H5N1 among a number of wild bird species. During the H5N1 outbreak in winter 2005/2006, pochards were either ranked first or second among wild bird species in terms of the number of birds found infected in France, Germany, and Switzerland  virus subtypes , and thetzerland . The spetzerland , the distzerland . The vertzerland  and theitzerland  are of aBased on ring recovery data, In this study, we use molecular data to describe the range-wide population structure of pochards. Our primary aim was to delineate the patterns of genetic differentiation between ducks on the breeding and wintering grounds across Eurasia, with a particular focus on assessing the previously postulated subdivisions. We further examine the congruence between nuclear and mitochondrial markers with different modes of inheritance to investigate sex-specific differences in the rates of gene flow . These aN≥ 9 for mitochondrial and/or nuclear markers were defined as population samples and included in all population genetic analyses while samples from other localities were included in the phylogeographic analyses only. Samples collected between June and August were considered to be from breeding populations, samples collected between November and February from wintering populations. For the breeding season, we obtained seven population samples plus smaller samples from eight additional locations. On the wintering grounds, population samples were obtained from 12 sites and additional samples from another four sites , and Germany . In total, 345 samples were available from 34 localities in 16 countries . Localitur sites . Three sur sites . Genomicur sites .ctr), including most of the hypervariable region (HVR) I, was amplified using the primers M1 , 1 μl MgCl2 (25 mM), 1 μl of each primer (10 pmol/μl), 2.5 μl 10× buffer (containing 1.5 mM MgCl2), and 0.2 μl Taq polymerase  and 2 μl template DNA (20–100 ng/μl). The PCR amplification profile included an initial denaturation step of 93°C for 2 min, followed by 35 cycles of 93°C for 30 sec, 56°C for 30 sec, and 72°C for 1 min, and a final extension step of 72°C for 7 min. PCR products were purified with the GenElute™ PCR clean-up kit (Sigma) according to the manufacturer's protocol. Sequencing reactions were carried out in a volume of 10 μl with the Terminator Ready Reaction Mix “Big Dye”  according to the guidelines of the manufacturer. The cycling protocol was as follows: 50 sec at 96°C, followed by 35 cycles of 10 sec at 96°C, 10 sec at 50°C, and 4 min and 30 sec at 60°C. The products were purified using sodium acetate precipitation, and were separated and detected on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems).A fragment of the mitochondrial control region  are a known issue in some diving ducks, including the genus Aythya , and mayAythya . Here, wAythya . Second,Aythya . FinallyAll samples were screened at 14 autosomal microsatellite loci initially developed for related species . The 14 k), the number of haplotypes (NH), haplotype diversity (H), and nucleotide diversity (π) were calculated for each population sample using DnaSP v.5.0 (AR), number of alleles (NA), and observed (HO) and expected heterozygosities (HE) in each population sample with FSTAT v.2.9.3.2 . Further, haplotype networks were constructed based on a median-joining algorithm (http://www.fluxus-engineering.com). The phylogeographic structure at the nuclear microsatellite markers was investigated with principal coordinates analyses (PCoA) based on pairwise Euclidian distances between individual genotypes in GENALEX v.6.2  and geographical distance in kilometers between pairs of populations. The significance of the association was assessed based on 1000 permutations.All population genetic analyses were carried out separately for the seven breeding populations and the 12 wintering populations. Analyses of molecular variance AMOVA;  implemenei model  and for K) was varied between 1 and 7 for breeding and 1 and 12 for wintering ducks, respectively, and we carried out 10 independent runs per K. The most likely K was assessed as suggested by We also used individual-based clustering approaches for the microsatellite data to investigate the genetic structure among breeding and wintering ducks. For Bayesian analyses with STRUCTURE v.2.3.1 , we usedFST = 0.001, 0.0025, 0.005, 0.01, 0.02, and 0.025) with samples sizes, numbers of markers, and allele frequencies corresponding to the empirical data. Statistical power was defined as the proportion of times the null hypothesis of equal allele frequencies across populations was rejected using a chi-square test or a Fisher's exact test.To assess the statistical power of our microsatellite markers to detect given levels of population differentiation, we performed power analyses in POWSIM v.4.0 . We genek) was similar in breeding and wintering populations .Sequences of 601 bp from the mitochondrial control region were obtained for 274 individuals, and 143 haplotypes were identified based on 80 polymorphic sites. Forty-seven haplotypes were shared by two to 36 individuals and the remaining 96 were singletons. The average number of pairwise nucleotide differences . There was no evidence of genotypic disequilibrium after Bonferroni correction. Significant heterozygote deficits were observed in 17 of 228 locus-specific tests with locus Caud13 being affected six times . For mtDNA, five of 21 pairwise comparisons between populations were significant (ST) increased with geographical distance between sampling locations . Based on the microsatellite markers, we obtained a global FST estimate of 0.018 (P < 0.0001), and five of 21 pairwise FST values were significant .The population-based analyses provided no evidence of major migratory divides across Eurasia. The four geographical groups  did not explain a significant proportion of the genetic variation for mtDNA or microsatellites . The varnificant , and thenificant . These cnificant . Again, Individual-based clustering approaches provided no evidence of genetic substructuring among ducks from breeding populations. STRUCTURE analyses showed highest support for one genetic cluster across Eurasia . DAPC inP > 0.15; ST of 0.02 (P = 0.0156), and two of 66 pairwise comparisons were significantly different from zero . For the microsatellite data, the overall population structure was very low but statistically significant , while none of the pairwise FST values were significant . No evidence of genetic substructure was detected by STRUCTURE  to investigate its movement patterns and the specific factors causing restricted gene flow between some of the breeding populations in more detail. Given that individuals are capable of traveling thousands of kilometers during annual migration, we would not expect physical restrictions to limit dispersal between breeding grounds. Indeed, the apparent absence of an isolation by distance (IBD) pattern between populations across Eurasia may suggest that dispersal occurs at least partially over very large distances as IBD can only build up if dispersal is spatially restricted . UnfortuIt is worth noting that IBD in pochard may not have built up because populations are not at mutation–drift equilibrium, for example, because of a recent range expansion of the species. At a local scale, such an expansion has occurred. During the last centuries, changes in the availability of suitable water bodies and food sources have led to a westward extension of the species’ breeding range into Central and Western Europe  However,N = 18), compared to 33% of the males . Along similar lines, In this context, attention should be paid to potential sex differences in colonization and dispersal patterns of pochard given the higher level of genetic differentiation among breeding grounds in maternally inherited mtDNA than in biparentally inherited microsatellite markers . Of courFST values were observed between European and Asian populations (Pochards sampled on the disjunct wintering grounds in Europe, the Caspian Sea, and eastern Asia show remarkably little genetic differentiation given the large geographical distances between them. Only population-based analyses including the wintering grounds in Asia revealed very weak signals of genetic subdivision. The latter result—taken alone—could be interpreted as reflecting a subdivision at least between the wintering grounds in Europe and East Asia at the western and eastern edges of the distribution range . Indeed,ulations . More imulations  demonstrGiven the very high levels of genetic diversity and relatively low genetic structure, it is not surprising that an aggregation of individuals from multiple breeding populations does not produce a classical population genetics signal for admixture among strongly subdivided populations such as elevated diversity or deviations from Hardy–Weinberg expectations (e.g., Wahlund effect; Extensive movements within and/or between winters are also the likely cause for the apparent genetic homogeneity among European wintering grounds of pochard. A subdivision was earlier proposed between a northwestern population wintering around the North Sea and a southeastern population wintering in central Europe and around the Mediterranean and Black Seas . HoweverThe absence of a clear association between genetic variation and geography and, in particular, the apparent mixing of ducks on wintering grounds imposes strong limitations on further analyses of the transmission and spread of pathogens of pochard. H5N1-positive individuals from European wintering grounds shared mtDNA haplotypes with ducks from several continental regions, and genetic differentiation was insufficient to use individual-based assignment methods to trace the geographic origin of pochard with highly variable microsatellite genotypes. However, complete panmixia is apparently prevented by low levels of breeding philopatry, which indicates that even higher genetic resolution might allow the identification of the large-scale region of origin of individuals gathering on wintering grounds. Such an analysis would require hundreds or thousands of markers distributed across the entire genome, as recently exemplified in humans, which also show globally low genetic population structure see . AdditioIt must be noted, however, that direct links between the movement of individuals or populations of pochard and the transmission and spread of HPAI H5N1 will be difficult to establish without considerably improved knowledge on the natural host spectrum, infection pathways, and the consequences of infection. H5N1-positive individuals were repeatedly found among pochard wintering in Europe but infections have also been detected in a number of other waterfowl species . Six pubContinent-wide transport of HPAI by individual birds may be rather unlikely but transfer in a stepping-stone fashion through successively infected birds appears nevertheless possible . In poch


Biological invasions are constantly gaining recognition as a significant component of global change. The Mediterranean fruit fly (medfly) constitutes an ideal model species for the study of biological invasions due to its (1) almost cosmopolitan geographic distribution, (2) huge economic importance, and (3) well-documented invasion history. Under a common garden experimental set up, we tested the hypothesis that medfly populations obtained from six global regions  have diverged in important immature life-history traits such as preadult survival and developmental times. We also tested the hypothesis that medfly populations from the above regions exhibit different population growth rates. For this purpose, data on the life history of immatures were combined with adult survival and reproduction data derived from an earlier study in order to calculate population parameters for the above six populations. Our results clearly show that medfly populations worldwide exhibit significant differences in preadult survival, developmental rates of immatures and important population parameters such as the intrinsic rate of increase. Therefore, geographically isolated medfly populations may share different invasion potential, since population growth rates could influence basic population processes that operate mostly during the last two stages of an invasion event, such as establishment and spread. Our findings provide valuable information for designing population suppression measures and managing invasiveness of medfly populations worldwide. Biological invasions represent one of the major components of global change . GlobaliDespite their negative effects, biological invasions offer an excellent opportunity for evolutionary studies. Invading populations of alien species are often subjected to different selective regimes than those in their native range. Under new abiotic and biotic conditions newcomers must survive, breed successfully, compete with other native species of the recipient community, avoid natural enemies and predators, and finally promote further survival and reproduction by expanding their range to suitable habitats . EvolutiCeratitis capitata (Wiedeman) , iedeman)  holds aniedeman) . C. capisiveness . Larval siveness , 1991. Osiveness . MedflieTaking into consideration these features that ensure biological success and adaptation in ecologically diverse habitats, it is not surprising that medfly is considered as a global invader that today exhibits an almost cosmopolitan geographic distribution. The genetic structure and affinity among medfly populations worldwide has been investigated in a series of detailed studies conducted during the past 20 years . DespiteIt is clear that besides its great contribution to aging and demographic research , medfly The experiments were conducted in the laboratory of Entomology and Agricultural Zoology at the University of Thessaly during summer 2005–autumn 2008, at 25 ± 1°C, 65 ± 5% R.H., and a photoperiod of L14:D10 with photophase starting at 0700 h. Light was provided by daylight fluorescent tubes and by natural light from four windows with the intensity inside the test cages ranging from 1500 to 2000 Lux.Prunus persica), (2) Africa , (3) Mediterranean , (4) Central America , (5) Pacific , and (6) South America . Details on the climatic conditions prevailing in the above regions are given in 1 generation for the comparison among medfly populations. Rearing of wild flies was accomplished by keeping adults, after emergence, in groups of about 100 individuals in wooden, (30 ´ 30 ´ 30 cm), wire-screened cages provided with water and a standard adult diet consisting of a mixture of yeast hydrolysate, sugar, and water (YS) at 4:1:5 ratio, respectively. Females were allowed to oviposit on 5-cm-diameter hollow, plastic hemispheres of red color (domes) that were artificially punctured with 40–50 evenly distributed holes on their surface. Eggs were deposited on the inner surface of the dome. Each dome was fitted in a 5-cm-diameter hole made on the cover of a 5.5-cm-diameter plastic petri dish. Water was placed in the base of the petri dish in order to maintain humidity levels beneath the dome adequate enough for female oviposition  Extra–Mediterranean  on an artificial diet  in orderAdults obtained from the above immatures were used to determine the survival and fecundity patterns of the above six medfly populations . These dt) = λ0(t) eXi)ηi ( (1) (i(Xi) = β1population1i+β2population2i+β3population3i+β4population4i+β5population5i (1a) is the linear component of the model, λ(t) denotes the probability of an individual reaching the next stage at any given time conditional on the observed covariates and λ0(t) the respective probability for the individual with baseline characteristics. All covariates are indicator functions with population1i, population2i, population3i, population4i, and population5i = 1 when subject originated from Guatemala, Greece, Brazil, Portugal, and Hawaii, respectively (else 0), so that Kenya forms the baseline. The covariate effects on egg, larval, pupal, and egg to adult developmental periods are measured by the beta coefficients in the linear component of the model. A c2 test was conducted to analyze the survival rates of immature stages with the application of the Bonferoni correction for multiple per two comparisons (http://www.r-project.org).Since normality tests failed for all duration of immature stages variables (Kolmogorov–Smirnov test used), survival analysis methodology was followed for all time to event data. Kaplan–Meier estimators of embryonic (egg), larval, pupal, and egg to adult developmental periods were calculated for each population. Pairwise comparisons of the respective periods between populations were conducted using the log-rank (Mantel–Cox) test. Effects of population on the duration of immature stages were assessed using the Cox proportional hazards model, λ((Xi) (1) . The estparisons . Confideparisons . Data anSignificant differences in immature survival were observed among the six populations tested for all stages apart from the pupal one . Egg to The differences among populations in the duration of preadult stages were significant as well . For exa2 = 371.8, df = 5, P < 0.001) (see Appendix S1 in supporting information), larval  (see Appendix S2), pupal  (see Appendix S3), and egg to adult  (The estimation of beta coefficients of the model (1a) shows a significant effect of population on the duration of egg (c< 0.001)  developm< 0.001)  denote tData on the life-history traits of immatures were combined with earlier ones on adult demography of the above six populations  to obtaiOur results demonstrate that medfly populations from six global regions exhibit differences in important life-history traits such as preadult survival and duration of immature stages. This result combined with differences in adult survival and reproductive schedule  account Paparge aegeria, the emergence of females in a very limited time window combined with nonoverlapping generations select for shorter immature development of males and protandry, whereas the reverse was observed for a population originating from Madeira, Portugal, probably due to overlapping generations  arrival, (b) establishment, and (c) spread . During Different growth rates among invading populations may also affect their spread during the last phase of an invasion. Invasive populations may follow different modes of spread. For example, in the case of continuous spread, a colonizing population moves along its front often following a simple diffusion model, with the intrinsic rate of population increase representing a critical factor regulating the rate of continuous range expansion into neighboring regions . As a coHowever, it should be borne in mind that a high intrinsic rate of increase alone cannot define the outcome of invasion . In an eOur results show that the population from Kenya exhibits the highest growth rate among the other populations tested, as indicated by its intrinsic rate of increase under stable conditions in the laboratory. Taking into consideration the loss of genetic variability, the establishment of an insect colony to the laboratory bears resemblance to the early phases of an invasion event. In the latter case, a significant loss of genetic variability may arise as the result of a limited number of invading individuals (founders), and the small population size in the following few generations . On the In conclusion, our results clearly demonstrate that, apart from divergence in adult survival and reproductive patterns , medfly


Peripheral blood leucopenia and thrombocytopenia are the main manifestations in severe fever with thrombocytopenia syndrome (SFTS) patients. However, the underlying causes are poorly understood. Therefore, we aimed to investigate cytology of bone marrow samples collected from SFTS patients. 10 SFTS patients were identified by typical clinical manifestations, detection of peripheral blood leucopenia and thrombocytopenia, and nucleic acid-based detection of the newly identified bunyavirus. SFTS patients, along with 10 participants with acute aplastic anemia and 10 healthy volunteers were enrolled in this study after written informed consent to undergo bone marrow cytological examination.We observed similar bone marrow properties in SFTS patients and healthy volunteers, significantly different from the characteristics observed in acute aplastic anemia patients.Similarities between bone marrow samples collected from SFTS patients and healthy volunteers suggest that peripheral blood leucopenia and thrombocytopenia do not result from bone marrow cell plasticity.  Recently, patients presenting with severe fever, digestive and respiratory symptoms accompanied by laboratory test results indicative of thrombocytopenia and leucopenia, were identified in rural regions of China, with some dying from multiple organ failure. This disease was designated severe fever with thrombocytopenia syndrome (SFTS) based on its clinical characteristics. In most cases, patients’ white blood cell and platelet counts were progressively reduced. However, administration of bone marrow-stimulating medications was seldom efficacious, while supplementation with exogenous platelets only temporarily maintained platelet levels. In an effort to unveil the mechanisms underlying the development of thrombocytopenia and leucopenia in SFTS patients, we compared these patients to acute aplastic anemia patients and healthy individuals for cytological properties of bone marrow samples.Whole blood and bone marrow samples from 10 SFTS patients (6 men and 4 women aged 30–67 [48.7 ± 11.2]) were obtained from the Departments of Infectious Disease and Hematology of Qilu Hospital, Shandong University and Jinan Infectious Diseases Hospital. Baseline characteristics and clinical outcomes are shown in This study, approved by the ethics committee of Shandong University, complied with the Declaration of Helsinki and Chinese law. All patients and healthy volunteers involved in this study provided written informed consents, signing their names in a document sent to the ethics committee of Shandong University.The Guide for Prevention and Treatment of SFTS published by the Ministry of Health of the People’s Republic of China in September 2010 was used to define the diagnostic criteria [bunyavirus nucleic acids. All blood samples were transported immediately upon collection to the Virus Laboratory of the Center for Disease Control for laboratory tests.Methodologies described by Espy  and Mack2, with just sufficient thickness to cover the slides. After samples were dried for 30 minutes at room temperature, Wright staining was performed, and stained slides microscopically evaluated by a trained technician. Bone marrow biopsies were collected at the same site, about 2-3 mm×10-12 mm marrow tissue was extracted and placed into a tube filled with 3-4 ml of Bouin solution for fixation. Then, tissues were dehydrated with high percentage ethanol and embedded into plastics (Hemapun 865 plastics). Sections were obtained with a microtome at a thickness of 3µm and slides submitted to Giemsa and Hematoxylin and eosin (H-E) stainings. Finally, sections were observed under light microscopy for hematopoiesis content, presence of fat and fibers, shape and number of marrow blood cells. All 10 SFTS patients agreed for bone marrow collection at the peak of illness (about 4-5 days after disease onset), but only 4 patients allowed bone marrow biopsies, the other 6 or their families being reluctant, worried about bleeding of the biopsy site. Therefore, 4 bone marrow biopsies were carried out in healthy volunteers for controls. All bone marrow samples were collected aseptically at bedside with the posterior superior iliac spine chosen as biopsy site. Bone marrow smear (0.3 mL) were aspirated and spread uniformly on 6 slides, each 2×3 cmThe statistical analysis of baseline characteristics of the 10 SFTS patients in comparison with volunteers and acute aplastic anemia patients are shown in P >0.10). Only 1.6 ± 0.6 megakaryocytes were observed per slide in acute aplastic anemia patients, significantly lower compared with patients SFTS . Representative micrographs of marrow cytology for the three groups are shown in Erythrocytes, megakaryocytes, and lymphocytes in bone marrow collected from the 10 SFTS patients displayed normal cell morphology, similar to healthy individuals. 12.3 ± 3.2 and 11.6 ± 4.2 megakaryocytes were counted per slide in samples from SFTS patients and healthy volunteers, respectively, a non- significant difference between these groups  was thought to be the etiological agent, and ticks the vectors. However, antibiotics effective against Rickettsia (such as tetracycline) were found not efficacious in the treatment of this disease. In 2011, after numerous studies, experts from the Institute of Virology of the Chinese Academy of Medical Sciences identified the pathogen as a novel virus, recently named bunyavirus, of which ticks appear to be the vectors [SFTS is a newly discovered disease with many unknown factors regarding pathogenesis and pathophysiology. Initially, SFTS was designated as human granulocytic anaplasmosis by clinicians.  vectors . SFTS is vectors ,6.  Clinically, it is believed that the main causes of peripheral blood thrombocytopenia and leucopenia include dysfunction of bone marrow regeneration, increased damage to the peripheral organs (such as spleen), and appearance of anti-platelet antibodies in the circulation. However, our data showed that bone marrow cytology was normal in SFTS patients, indicating an optimal bone marrow cell regenerative capacity. Thus, it is tempting to speculate that peripheral blood thrombocytopenia and leucopenia in SFTS patients result from increased peripheral organ damage or circulating anti-platelet antibodies. SFTS symptoms are markedly different from the manifestations of hemorrhagic fever with renal syndrome, caused by hantavirus (a bunyavirus subspecies), where thrombocytopenia in peripheral blood is a major phenotype, but with increased instead of reduced total white blood cell count . The dif


Theoretical models predict that a cost is necessary to guarantee honesty in begging displays given by offspring to solicit food from their parents. There is evidence for begging costs in the form of a reduced growth rate and immunocompetence. Moreover, begging implies vigorous physical activity and attentiveness, which should increase metabolism and thus the releasing of pro-oxidant substances. Consequently, we predict that soliciting offspring incur a cost in terms of oxidative stress, and growth rate and immune response (processes that generate pro-oxidants substances) are reduced in order to maintain oxidative balance.Pica pica) nestlings incur a cost in terms of oxidative stress when experimentally forced to beg intensively, and whether oxidative balance is maintained by reducing growth rate and immune response. Our results show that begging provokes oxidative stress, and that nestlings begging for longer bouts reduce growth and immune response, thereby maintaining their oxidative status.We test whether magpie , oxidative stress, and life-history traits directly linked to viability. Evolutionary theory hypothesizes that many animal signals have to be costly in order to reliably indicate signaler quality Nestlings may incur different types of direct costs when begging intensively. First, loud begging calls may attract eavesdropping predators to the nest and nestling jostling may increase brood conspicuousness Begging usually involves vigorous posturing and calling Pica pica) nestlings into begging for long bouts during three consecutive days at the laboratory , while their control nestmates were kept begging at a much shorter rate  for the same amount of food. Magpie nestlings show reduced growth and immunocompetence when begging at high rates (Nestlings begging intensively may reduce their growth rate  and immuClamator glandarius) reduced sample size to 12 available unparasitized broods of an appropriate age. We used 32 nestlings  when they were 9 d old, i.e. two days before the age at which they grow at the highest rate ad libitum. Nestlings were maintained in the laboratory for three days, and when the experiment ended, nestlings were fed ad libitum again and returned back to their nests during the morning. No nestling died or suffered damage during the study, and parents accepted all nestlings returned back to their nests.The study was carried out during the spring of 2010, with a population of European magpies located at Santa Fe and Chimeneas (SE Spain). The study area is formed by a mix of farmlands, mainly cereals, with scattered olive and almond trees, where the magpies nest. Nests were inspected regularly to determine the exact date of hatching (day 0). We located 42 active nests with complete clutches, but high rates of nest destruction by local people, natural predation, and parasitism by great spotted cuckoos (n = 16) or a low begging  treatment. Four nests contributed two pairs to the experiment. During the three days of the experiment, each nestling was maintained isolated in a cloth cup simulating a nest, at a constant temperature of ca. 36°C. While resting, nestlings were covered by a duster, simulating brooding by the mother, which precluded nestlings from begging between trials. During each feeding, for the three days of the experiment, nestlings were stimulated to beg by using a human word (“toma”) at which nestlings were previously trained to respond the day before the experiment began. Low begging (LB) nestlings were fed immediately after gaping, while HB nestlings were stimulated to beg for 1 min before being fed. Therefore, experimental manipulation caused HB nestlings to beg for considerably longer bouts than their LB nestmates. Considering that magpie nestlings beg for 7–18 s/hour on average r = 0.78; P<0.001), suggesting that estimates were repeatable. Technical difficulties with recording three LB nestlings reduced sample size for the variable “time begging” to 13 LB nestlings.We randomly assigned one member of each pair of nestmates of a similar body mass to either a high begging  and enriched with vitamins A, D3, and E, calcium and phosphorus. Feces excreted by nestlings were weighed. Mass gained was estimated as the difference in body mass between the first and the last day of the experiment.Each morning, nestlings were weighed before being fed with a digital balance (accuracy 0.01 g) and the mass of the first fecal sac discounted, to calculate body mass. We estimated the food to be ingested by nestlings during each experimental day according to their mass, following the allometric relationship between daily food consumed and daily growth We also measured how the experimental treatment affected the ability to mount a general inflammatory response. The third day of the experiment, at 20∶00 h in the evening, we injected into the left patagium of each chick 0.4 mg of phytohaemagglutinin  diluted in 0.08 ml of isotonic phosphate buffer g during 10 min, and plasma was separated from red cells. Red blood cells were cleaned with 9 g/L NaCl solution and centrifuged again, removing the supernatant. Then, samples of erythrocytes were diluted in distilled water (proportion of 1∶4), provoking cell lysis, and the hemolysate was frozen at −80°C until the analyses. All enzymatic assays were carried out at 25±0.5°C using a PowerWavex microplate scanning spectrophotometer  in duplicate in 96-well microplates . We measured the activity of Superoxide dismutase (SOD), Glutathione peroxidase (GPX), and Glutathione reductase (GR) following standardized techniques  for concentration of MDA, soluble protein, and activity of GPX enzyme, and it was 0.71  for SOD and 0.78  for GR enzymes.When the experiment ended, we extracted approximately 200 µl of blood from the brachial vein during the last morning the chicks were in the lab. By taking blood samples after nocturnal fast, problems with effects of digestion on biochemical traits are avoided chniques . EnzymatFor statistical analyses, we performed Generalized Linear Mixed Effects Models of Restricted Maximum Likelihood (REML-GLMM) n = 16) and low begging  nestlings . Note that mass gained was negatively correlated with immune response .There were no significant differences in initial body mass, food ingested and feces mass excreted, between high begging  to be higher in HB (37.9±2.8 nmoles/ml) than in LB nestlings  At a first glance, there were not differences in levels of oxidative damage between HB and LB nestlings, but nestlings begging for longer reduced mass gained and immune response. When we controlled for mass gained and immune response, the positive correlation between begging time and oxidative stress emerged, which suggests that nestlings begging for longer reduced mass gain and immune response to avoid oxidative damage. Begging implies physical and neurological effort, which increases metabolism and thus RONS releasing On the other hand, nestlings might sustain high begging rates at low levels of oxidative stress by reducing other pro-oxidant components of life-history, such as growth and immune response. Both growing and mounting an immune response increase metabolic expenditure, contributing to RONS releasing. By reducing growth rate and immune response, nestlings may avoid immediate negative consequences of oxidative stress but, in turn, incurred other viability costs. Body size is an important determinant of survival in magpie nestlings and fledglings β = 0.24; F1, 13 = 5.70, P = 0.03). This result suggests that reduction of mass gained at day 1 was a mechanism aimed at maintaining oxidative balance. In addition, there is compelling evidence showing that activities that are metabolically demanding, such as reproduction or physical exercise, may impair immune function in birds Click here for additional data file.Table S1Model showing the effect of treatment on protein concentration.(PDF)Click here for additional data file.Table S2Model showing the effect of treatment on the concentration of active enzyme superoxide dismutase (SOD).(PDF)Click here for additional data file.Table S3Model showing the effect of treatment on the concentration of active enzyme glutathione peroxidase (GPX).(PDF)Click here for additional data file.Table S4Model showing the effect of treatment on the concentration of active enzyme gluthatione reductase (GR).(PDF)Click here for additional data file.Method S1Measurement of enzymatic activity.(PDF)Click here for additional data file.