Significant positive correlation also exists among activities of AGPase, SSS, GBSS, and SBE, except for GBSS-SBE. In summary, our study has revealed that the reduced conversion of sucrose to starch in the late grain filling stage is the main cause for the low kernel weight and total starch accumulation of the waxy wheat. The reduced conversion also appears to be a factor contributing to the lower grain yield of the waxy wheat.Waxy wheat has unique end-use properties; however, its production is limited due mainly to its low grain yield compared with non-waxy wheat. In order to increase its grain yield, it is critical to understand the eco-physiological differences in grain filling between the waxy and non-waxy wheat. In this study, two waxy wheat and two non-waxy wheat cultivars were used to investigate the differences in starch-associated enzymes processes, sucrose and starch dynamics, yield components, and the final grain yield. The results indicated that the mean total grain starch and amylose content, the average 1000-kernel weight and grain yield of the waxy wheat were lower than those of the non-waxy wheat at maturity. The amylose content was significantly and positively correlated with the activity of GBSS (r = 0.80,  Common wheat cultivars have three homeologous waxy genes, Wx-A1, Wx-B1 and Wx-D1, and waxy wheat mutants lack all three Wx proteins. The Wx proteins are also known as granule-bound starch synthases, which have a role in the synthesis of amylose5. However, due to low grain yield, the waxy wheat has not been widely cultivated in recent years. To improve the current situation, there is an urgent need for improved understanding of the differences in yield performance between the waxy and non-waxy wheat cultivars at the field level.With the growing in health-conscious consumers, there has been an increase in demand for high-quality wheat flour. The endosperm starch content and composition are two critical parameters for evaluating the quality of wheat flour. For conventionally cultivated wheat, namely non-waxy wheat, starch is a mixture of about 75% amylopectin and 25% amylose. To further improve the quality of wheat flour products such as reducing staling in flour products, keeping baking goods fresh for a longer period of time, and improving the palatability of noodles, the replacement of regular wheat flour with waxy wheat flour may provide a promising solution, as waxy wheat grains contain almost 100% amylopectin in their starch7, but also reflects the capacity of the sink tissues. Regarding the source-sink relationship, the photoassimilates produced in source tissues as leaf, stem and root were transported into the amyloplast in the form of sucrose, which is the main carbohydrate transported in higher plants. Sucrose is degraded to fructose and uridine diphosphate glucose (UDPG) which are the main precursors of starch synthesis by sucrose synthase (SUS: EC 2.4.1.13)9. The ability of the sink tissues to accept and convert photoassimilates can be affected by the sink strength, which is an important limiting factor to wheat grain yield, and the activity of SUS can be considered as an indicator of sink strength11.The endosperm starch content (approximate 70% of the dry weight) not only influences the grain weight and quality12, including ADPG pyrophosphorylase (AGPase: EC 2.7.7.27), starch branching enzyme (SBE: EC 2.4.1.18), starch debranching enzyme (DBE), granule bound starch synthase (GBSS: EC 2.4.1.21) and soluble starch synthase(SSS: EC 2.4.1.21). GBSS and SSS are the two forms of starch synthase14. For catalyzing the first unique step in starch synthesis, AGPase is the rate-limiting enzyme and is the most important determinant of seed sink strength16. Starch synthases catalyse the elongation of the linear glucan chains17, moreover the different genetic characterization assigns preferential functions for individual isoforms to synthesize amylose or amylopectin. It is generally known that in the endosperm, amylose is synthesized by AGPase and GBSS13. SBE that was formerly known as Q-enzyme plays an important role on amylopectin synthesis, which is the only plant enzyme that can introduce α-1,6-glucosidic linkages into α-polyglucans19.A coordinated series of enzyme-catalyzed reactions in wheat endosperm result in starch synthesisWx genes22. It is reported that the seed weight of inferior spikelets of rice can be improved by increasing the starch-synthesizing enzymes activities23. In this study, we compared two waxy wheat cultivars with two non-waxy wheat cultivars to investigate the starch content and accumulation, the activities of enzymes responsible for starch biosynthesis in developing grain, 1000-kernel weight and grain yield with the objective to better understand the differences between the wheat varieties and elucidate factors affecting the 1000-kernel weight and grain yield of the waxy wheat.Previous researches on waxy wheat have focused on the end-use quality, composition of the grain and the expression of the −1 organic matter, 100.01 mg kg−1 alkali hydrolysable N, 50.11 mg kg−1 Olsen-P, and 149.08 mg kg−1 exchangeable K at 0–20 cm soil depth in 2012, while 19.57 g kg−1 organic matter, 107.97 mg kg−1 alkali hydrolysable N, 33.92 mg kg−1 Olsen-P, and 124.23 mg kg−1 exchangeable K at 0–20 cm soil depth in 2013.Field experiments were conducted at the Agricultural Experiment Station 32°39′N, 119°25′E) of the Agricultural College of Yangzhou University in China during the winter wheat growing seasons in 2012–2013, and 2013–2014. The field site is situated in a humid subtropical climate zone, with average air temperature of 13 °C to 16 °C, total precipitation of 800 to 1200 mm, total sunshine of 2000 to 2600 h, and a frost-free season of 220 to 240 d. The meteorological data during the wheat growing seasons across the two study years, including temperature and precipitation are shown in Fig. 19°25′E oWx-A1, Wx-B1, Wx-C1 alleles based on a PCR test (data not shown)] and two non-waxy wheat cultivars Yangmai20 (Y20) and Yangfumai4 (YF4), .A total of four wheat cultivars were tested, including two types of waxy wheat cultivars Yangnuomai1 (YN1), Ningnuomai1 (NN1), [which lack functional −1, applied with the ratio 5:1:2:2 at four stages: pre-sowing, four-leaf stage, jointing stage and booting stage, respectively. Application rate of P2O5 (P) and K2O (K) were both 144 kg ha−1: half the amounts of P and K were applied before sowing and the remaining half were applied at the jointing stage. The seeds were sown at a planting density of 225 × 104 ha−1 with 30 cm row spacing on 3 November of 2012 and 28 October of 2013. Seedling number was maintained at 225 × 104 ha−1 by removing or adding seedlings at the three-leaf stage. Plants were harvested on 3 June of 2013 and 28 May of 2014. Weeds, insects and diseases were controlled as required to avoid yielding loss; other production management practices were based on those used by local farmers.All plots were supplied with 240 kg N ha24.At maturity, an area of 1.2 × 1 m (4 rows included) was harvested manually for the determination of the grain yield. Grain yield was adjusted to 13% moisture. Number of spikes was counted manually for two rows within 1 m. The number of kernels per spike was counted manually to calculate the mean value from 50 continuous spikes. For 1000-kernel weight, 1000 kernels were randomly selected and weighed. The average seed volume was determined via the displacement method26. Wheat grains that were marked flowering on the same day were first ground using a mortar, and the powder was then degreased twice with anhydrous ether. A 100 mg fraction of each sample was used to determine amylose and amylopectin contents. A calibration curve was derived using pure amylose from potato  and pure amylopectin from potato . The sum of amylose and amylopectin contents was designated as the total starch content.The total starch, amylose, and amylopectin contents were determined via the dual wavelength iodine binding method28. SSS and GBSS activities were assayed according to Nakamura and Cheng28. SBE activity was assayed according to Li et al.29.Twenty kernels weighed at different filling stages were tested. AGPase activity was assayed according to Nakamura and Cheng31. Ten dried spikes those were flowered on the same day were selected and killed at 105 °C for 30 min and then dried at 80 °C for measurements of grain weight and starch content. And then they were weighed, and then the dried endosperm was ground into powder for analysis. Samples of the powdered endosperm prepared as mentioned above (100 mg) were extracted with 8 mL of 80% ethanol at 80 °C for 30 min, followed by two extractions with 8 mL of 80% ethanol. The supernatants were combined and purified by 10 g activated carbon overnight, fixed to a constant volume at 50 ml, and purified by filtering. One-hundred microliters of 2 M sodium hydroxide was added to 0.9 ml filtrate, boiled at 100 °C for 10 min. The mixture was cooled to room temperature, then 1 ml of 0.1% resorcinol and 3 ml of 10 mol/L hydrochloric acid were added. The reaction mixture was boiled at 80 °C for 60 min, cooled to room temperature, and the sucrose content was determined by measuring OD at 500 nm.The sucrose content was measured by the resorcinol technique34.The SUS activity was assayed according to Rufty, Doehlert and WardlawP = 0.05. Pearson’s correlation analysis (SAS V.9.1) was performed to establish the relationships among all measured properties. The ANOVA (α = 0.05) was performed to identify significant differences between treatments, multiple comparisons between means (SAS V.9.1) were made using the least significant difference (LSD0.05) test if the F-tests were significant.All data were checked for normality based on the Kolmogorov–Smirnov test with SAS proc mixed (SAS V.9.1). The effects of year, cultivar and their interaction on test parameters were tested with year, cultivar as fixed effects, year as repeated measurements, and block as a random effect. Differences among least square means (LSMEANS) for all treatment pairs were tested by the LSD procedure at a significance level of The test found that grain sucrose content was significantly affected by cultivar, year and their interactions Fig. . Averageth–25th days after anthesis, but decreased after the 25th day after anthesis for both types of wheat  increased during early grain filling period and decreased during the late period after anthesis. The peak activities of the AGPase and GBSS occurred on the 25day Fig. . The AGPpy = 0.0072, pc < 0.0001), number of spikes  and the 1000-kernel weight ; however, the interaction between year and cultivar was not significant. For the grain yield, a significant cultivar effect was detected (pc < 0.0001), but not for year and year-cultivar interaction.The year and cultivar significantly affected the number of kernels per spike , while the amylose content did not significantly correlate with other enzyme activities except for GBSS  and sink activity as indicated by activities of the enzymes such as SUS and AGPase in endosperm, which is closely associated with carbohydrate utilization and storage41. Moreover, early studies have found that waxy wheats generally have higher sucrose content and lower transforming ability compared with non-waxy wheat42. The sucrose content is significantly correlated with the starch synthesis in the early period of grain filling (before 25th days after anthesis) in wheat43 and rice44. The SUS plays an important role in sucrose synthesis and degradation in plants, especially in wheat grains; It is generally believed that the main function of the SUS is to catalyze the degradation of sucrose in grains46. The decrease of SUS activity leads to lower rate of conversion from sucrose to starch and a high residual sucrose content43, and further resulting in a lower dry matter accumulation characteristic of rice grains48. In this study, we recorded significant differences in the sucrose content and SUS activity in grains among waxy and non-waxy wheat cultivars after anthesis. Throughout the grain filling, the grains of waxy wheat had higher sucrose content, but SUS activity in grains was different between the earlier and later stage after anthesis. Our results suggested that waxy wheat had higher sucrose degradation and bioavailability during early grain filling stages, but was lower at the later stage compared with non-waxy wheat. More sucrose and less grain starch synthesis ability could be characteristic of waxy wheat in comparison to non-waxy wheat especially in the late period after anthesis. These results were in agreement with some previous studies conducted in waxy42 and non-waxy46 wheats.The conversion of sucrose to starch in wheat grain was closely related to starch accumulation, for example, high starch accumulation was observed when the conversion of sucrose to starch was high49, which is the major factor influencing both grain yield and quality12. The first unique step of the starch synthesis is catalyzed by AGPase, which is considered to be the rate-limiting enzyme because of the positive correlation between the activity and starch accumulation rate51. In this study, AGPase activity increased at the early grain filling stage, leading to increased accumulation of the total starch, amylose and amylopectin, as well as their accumulation rate. We also found that the AGPase activity in waxy wheat was lower than that in non-waxy wheat in the whole grain filling period, resulting in lower total starch content in waxy wheat seeds compared with non-waxy wheat. At 30d after anthesis, the AGPase activity started to decrease, resulting in a reduction in the rate of starch, amylose and amylopectin accumulation. During the later grain filling period, the starch accumulation and accumulation rate of waxy wheat were lower than that of non-waxy wheat. Similar findings have also been reported in previous studies: in other crops higher AGPase activity resulted in higher crop starch biosynthesis and grain yield including maize52, rice53 and wheat54.Starch is synthesized and accumulated during the grain-filling process17. The absence of waxy protein resulting from waxy mutations in wheat and other cereals could lead to amylose-free starch in endosperm, with low activity of GBSS in waxy wheat55. GBSS activity determines amylose content in the endosperm of rice56 and wheat57. In this study, the GBSS activity of waxy wheat was significantly lower than that of non-waxy wheat throughout the grain filling period; the amylose accumulation and accumulation rate in waxy wheat were also lower than that in non-waxy wheat. These results indicated that waxy wheat had lower grain starch and amylose synthesis capacity compared with non-waxy wheat, particularly in the case of amylose. Moreover, GBSS is a critical component in the process of amylose formation58; some studies have also reported that reduction of amylose content and GBSS activity has been observed in many species that lack amylase and all of these specifically lack GBSSI activity14. GBSS is a critical enzyme in the process of amylose biosynthesis58 and its activity is closely associated with amylose content as observed in many species that lack amylase and GBSS, specifically the GBSSI activity14.GBSS is exclusively involved in the synthesis of amylose, where it catalyzes the extension of long glucans within the amylopectin fraction59. Amylose, amylopectin and total starch accumulation rate in wheat grains were significantly and positively correlated with activities of SBE, SSS and GBSS60, and a reduction in enzyme activity appears to be the main factor affecting decreased starch synthesis43. In this study, enzymatic activities of SSS and SBE were higher in waxy wheat than in non-waxy wheat, while amylopectin accumulation and amylopectin accumulation rate was significantly higher in waxy wheat compared with the non-waxy wheat during early grain filling stages. During the late grain filling stage, activities of both enzymes were lower, while the amylopectin accumulation and accumulation rate decreased more quickly in waxy wheat compared with non-waxy wheat. These results were similar to those of Tan et al.61 who reported that lower activities of AGPase, SSS, GBSS and SBE and the lower accumulation of amylose, amylopectin and starch in waxy wheat compared with non-waxy wheat and also in lower starch accumulation cultivar had lower enzyme activities in non-waxy wheat58.The SBE activity did not significantly influence the amylopectin content but significantly positively related to the amylopectin accumulation rate, it is postulated that SBE might not play an important role in the formation of amylopectin, but to its fine structure modification, it is stated in maize for the unique function of SBEI in modulating the branching pattern in normal starch by decreasing local clustering of amylopectin branch points62.The SSS and SBE play an important role in the formation of the branched structure of amylopectin molecules42, and also lower starch, amylose and amylopectin accumulation and accumulation rate than non-waxy wheat in the grain filling period61. It is possible that higher soluble sugar content and lower starch content were due to a low conversion efficiency and the absence of GBSS at the period of grain filling because these two factors have been shown to influence grain weight in waxy rice63 and in waxy wheat20. Higher activities of SBE, APGase, SSS and GBSS are generally attributed to increased kernel weight in wheat, while starch accumulation rate is significantly correlated with activities of SBE, AGPase, SSS and GBSS in both waxy wheat61 and non-waxy wheat41. Seed sink strength and seed yield would all be expected to be enhanced by increasing the AGPase activity16. The activities of AGPase, SSS, GBSS and SUS play important roles in determining individual grain weight in wheat by regulating starch synthesis in grain endosperm64. Further, many studies show that there is a coordinating action in starch biosynthesis among these enzymes60. The four important enzymes for starch biosynthesis played the important role but not the whole starch biosyntheses net.Waxy wheat was found to have lower starch content and higher sugar content than non-waxy wheatIn this study, grain yield appeared to be influenced by sucrose and starch factors. The total starch and amylose content at maturity in grains of waxy wheat were 13.29 and 88.27% lower respectively than those of non-waxy wheat. The 1000-kernel weight of waxy wheat was 7.05% lower than non-waxy wheat, and the grain yield of waxy wheat was 12.33% lower than that of non-waxy wheat. Reduced activities of AGPase, SSS, GBSS and SBE and a lower accumulation of amylose, amylopectin and starch were also observed during the later stage of grain filling in waxy wheat compared with non-waxy wheat. The amylose content was significantly and positively correlated with GBSS activity, while amylose was significantly and positively correlated with the 1000-kernel weight. The total starch content and amylopectin content were significantly and positively correlated with AGPase, SSS and SBE activities. Among the activities of AGPase, SSS, GBSS and SBE, there were significant correlations, except between the activities of GBSS and SBE.Our study suggests that low AGPase activity could be the cause of low total starch content in waxy wheat grain, and low GBSS activity could be the contributing factor to insufficient amylose content. During the early grain filling period, no significant difference was found in sink size between the waxy and non-waxy wheat, while higher sucrose degradation and bioavailability and activities of SSS and SBE in grains of waxy wheat were found. Low activities of SSS and SBE in waxy wheat are likely the cause of low amylopectin accumulation in the later stage of grain filling. We speculate that the four enzymes, AGPase, SSS, GBSS and SBE, may have a complementary relationship with regards to starch biosynthesis; thus, the absence of GBSS can reduce the activity of the other three enzymes, leading to low total starch content in grains of waxy wheat. The low activity of SUS during later period of grain filling may have caused the reduced degradation of sucrose and the absence of the precursor of starch in grains of waxy wheat. It appears that weakened starch synthesis in the later stage of grain filling of waxy wheat is likely to be the main cause of low kernel weight and total starch accumulation, which ultimately leads to lower grain yield in waxy wheat.


In natural forests, tree leaf surfaces host diverse bacterial communities whose structure and composition are primarily driven by host species identity. Tree leaf bacterial diversity has also been shown to influence tree community productivity, a key function of terrestrial ecosystems. However, most urban microbiome studies have focused on the built environment, improving our understanding of indoor microbial communities but leaving much to be understood, especially in the nonbuilt microbiome. Here, we provide the first multiple-species comparison of tree phyllosphere bacterial structures and diversity along a gradient of urban intensity. We demonstrate that urban trees possess characteristic bacterial communities that differ from those seen with trees in nonurban environments, with microbial community structure on trees influenced by host species identity but also by the gradient of urban intensity and by the degree of isolation from other trees. Our results suggest that feedback between human activity and plant microbiomes could shape urban microbiomes. Alphaproteobacteria. Our work in the urban environment here reveals that the structures of leaf bacterial communities differ along the gradient of urban intensity. The diversity of phyllosphere microbial communities increases at higher urban intensity, also displaying a greater number and variety of associated indicator taxa than the low and medium urban gradient sites. In conclusion, we find that urban environments influence tree bacterial community composition, and our results suggest that feedback between human activity and plant microbiomes could shape urban microbiomes.Tree leaf-associated microbiota have been studied in natural ecosystems but less so in urban settings, where anthropogenic pressures on trees could impact microbial communities and modify their interaction with their hosts. Additionally, trees act as vectors spreading bacterial cells in the air in urban environments due to the density of microbial cells on aerial plant surfaces. Characterizing tree leaf bacterial communities along an urban gradient is thus key to understand the impact of anthropogenic pressures on urban tree-bacterium interactions and on the overall urban microbiome. In this study, we aimed (i) to characterize phyllosphere bacterial communities of seven tree species in urban environments and (ii) to describe the changes in tree phyllosphere bacterial community structure and diversity along a gradient of increasing urban intensity and at two degrees of tree isolation. Our results indicate that, as anthropogenic pressures increase, urban leaf bacterial communities show a reduction in the abundance of the dominant class in the natural plant microbiome, the IMPORTANCE In natural forests, tree leaf surfaces host diverse bacterial communities whose structure and composition are primarily driven by host species identity. Tree leaf bacterial diversity has also been shown to influence tree community productivity, a key function of terrestrial ecosystems. However, most urban microbiome studies have focused on the built environment, improving our understanding of indoor microbial communities but leaving much to be understood, especially in the nonbuilt microbiome. Here, we provide the first multiple-species comparison of tree phyllosphere bacterial structures and diversity along a gradient of urban intensity. We demonstrate that urban trees possess characteristic bacterial communities that differ from those seen with trees in nonurban environments, with microbial community structure on trees influenced by host species identity but also by the gradient of urban intensity and by the degree of isolation from other trees. Our results suggest that feedback between human activity and plant microbiomes could shape urban microbiomes. While the human population in urban centers is estimated to increase by 2 to 4 billion this century , the foc12–8 km2 across the globe (––Hedera sp. and reference Quercus macrocarpa), leaving much to be learned on how the plant microbiome changes with increasing anthropogenic pressures. In this report, we focus on tree phyllosphere bacterial communities along a gradient of urban intensity to quantify the similarities and differences in community structure in the urban plant microbiome.The phyllosphere, defined as the leaf surfaces of plants, is estimated to have a surface area of 4 × 10he globe  and thushe globe , 17. In he globe . Recent he globe –23 and te globe ––28. HoweIn the urban ecology literature, many different study-specific methodologies have been employed to estimate the intensity of human influence in an area, reducing the potential to compare results between experiments. In an attempt to standardize our work, we use the well-described index by Sanderson et al.  which haThe urban environment differs strikingly from the natural forest environment mainly through an increase in the biotic and abiotic stresses caused directly and indirectly by anthropogenic activities. The increase in anthropogenic pressures in urban areas reduces tree fitness and longevity  and coul36–52–To improve our understanding of the urban tree microbiome, we aimed (i) to characterize the bacterial communities present in tree phyllosphere bacterial communities of the urban environment and (ii) to describe the changes in tree phyllosphere bacterial community structure and diversity along a gradient of increasing urban intensity and at two degrees of tree isolation. Our hypotheses are (i) that the increasing urban intensity influences the abundance of the main taxonomical groups of bacteria usually described in the natural temperate forest phyllosphere literature and (ii) that the amount of stresses on trees imposed by higher anthropogenic pressures in urban agglomerations  impacts phyllosphere bacterial community structure and reduces its diversity from low urban intensity to higher urban intensity settings. While previous urban microbiome studies have focused on air and built environment microorganisms  at three sites in Montreal, Canada, along a gradient of increasing anthropogenic pressures. This urban intensity gradient was based on a composite index of human influence  as empl10.1128/mSystems.00087-17.1TABLE S1 TABLE S1, DOCX file, 0.02 MB.Description of the three study sites sampled during summer of 2014. Download Copyright © 2017 Laforest-Lapointe et al.2017Laforest-Lapointe et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Our data set contained 8,671 operational taxonomic units (OTUs) with 3,634 to 32,060 sequences per sample for a total of 1,755,757 quality sequences from 126 samples. We rarefied the samples to 3,000 sequences each, with 18 samples excluded due to insufficient sequence reads as a result of extraction or sequencing errors.Alphaproteobacteria, averaging 40.8%, 39.4%, and 31.9% of sequences in communities from settings of low, medium, and high urban intensity, respectively. The five most abundant bacterial classes in the urban phyllosphere were Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, Cytophagia, and Actinobacteria. Only the members of the class Alphaproteobacteria displayed a significant change in relative abundance along the urban gradient , with differences of 3.4% and 2.0% comparing the high-intensity sites to the low-intensity and medium-intensity sites, respectively   .Acer platanoides, Acer rubrum, Acer saccharum, Celtis occidentalis, Fraxinus americana, Fraxinus pennsylvanica, and Picea glauca), tree isolation (park or street), and urban intensity . Because the two models yielded highly similar results in terms of significant variables and explanatory power, here we present only the results of analyses based on Bray-Curtis dissimilarities. We illustrated bacterial community taxonomic structure patterns by performing a nonmetric multidimensional scaling (NMDS) ordination of Bray-Curtis dissimilarities  and tree isolation  were both weaker but significant drivers of leaf community structure. All second-level interactions were significant, the strongest being the interaction between urban intensity and host species identity , and the third-level interaction was also significant . At the class taxonomic level, we found multiple significant correlations among the relative abundances of various bacterial groups with the two axes of the NMDS , followed by C. occidentalis , F. americana , and F. pennsylvanica . The effect of urban intensity was not significant for A. saccharum  and was only marginally significant for P. glauca . Since host species identity was the main driver of leaf bacterial community structure across the urban gradient, we built a model accounting for phylogenetic relatedness between host tree species to identify the explanatory power of different phylogenetic levels. The angiosperm/gymnosperm level explained 4.5% of the total variation in phyllosphere bacterial community structure, whereas the genus and species levels explained, respectively, 7.2% and 8.3% of the variation.We quantified the relative influences of multiple drivers on leaf bacterial community taxonomic and phylogenetic structure by conducting a permutational multivariate analysis of variance (PERMANOVA ) on Braylarities . The strthe NMDS , includis models . Urban iF = 7.067, P = 0.001) and host species  as well as their interaction . Comparing the leaf bacterial alpha-diversity levels across sites, the highest level of urban intensity exhibited greater leaf bacterial alpha-diversity  than the low urban intensity  and the medium urban intensity . Since the interaction between the urban intensity and host species identity was significant, we computed per-host species alpha-diversity indices (Chao 1 and Shannon diversity), as well as community evenness (Shannon evenness) . We compared the strengths of the variables in the best model by an ANOVA, which included urban intensity (venness) . All speP value of <0.05 were classified as indicator species , 9 associated with the medium level , and 37 indicators associated with the high level . In addition, we built a three-axis ternary plot  have been found by Smets et al.  and leaf microbial communities. Future experiments developing a “germfree” or “microbiome-controlled” plant growth system in greenhouses will allow researchers to separate the relative influences of the environment and the host microbiome.Increasing levels of anthropogenic pressures, including land use changes, biogeochemical changes, global warming, and exotic species invasion, cause an augmentation of tree stress and a corresponding diminution of longevity and productivity –54. Here–per se is different from what is usually found in the natural environment as described in previous work in the natural temperate forest  represented low urban intensity; Ahuntsic  represented medium intensity; and Mont-Royal  represented high intensity . We asseAll samples were acquired on 31 July 2014. For each randomly chosen mature tree (at least 20 cm in diameter), we clipped 50 to 100 g of shade leaves at midcanopy height (1 to 2 m above the bottom of the tree’s canopy) into sterile roll bags with surface-sterilized shears. For bacterial community collection and amplification, we used the protocols described by Kembel et al. (Samples were amplified using a one-step PCR step and normalized with primers designed to attach a 12-bp barcode and Illumina adaptor sequence to the fragments during PCR . For allWe processed the raw sequence data with PEAR  and QIIMTo exclude any spurious OTUs that might have been created by PCR or sequencing errors, we filtered the OTUs to remove those that were represented by fewer than 5 sequences. Rarefactions and analyses were repeated 100 times and showed no qualitative differences across iterations. Therefore, we present the result of a single random iteration. We performed analyses with the ape , picantehttps://figshare.com/s/07342fc588afdf8d1499 for the R code; https://figshare.com/s/16b8c237fe9b46d3da9b for the metadata; and, finally, https://figshare.com/s/af205b286c6f88f4b1f1 for the 16S RNA sequences.The data sets supporting the conclusions of this article are available in the Urban Tree phyllosphere Microbiome project repository on Figshare at


AbstractGymnocalyciumkieslingiisubsp.castaneum (Ferrari) Slaba was analyzed. Fifteen plants were selected and from each plant one areole from the basal, middle and upper part of the plant body was sampled. A scanning electron microscopy was used for spine surface description and a light microscopy for measurements of spine width, thickness, cross-section area, fiber diameter and fiber cell wall thickness. The spine surface was more visible and damaged less in the upper part of the plant body than in the basal part. Large spine and fiber differences were found between upper and lower parts of the plant body, but also within single areoles. In general, the examined traits in the upper part had by 8–17% higher values than in the lower parts. The variation of spine and fiber traits within areoles was lower than the differences between individuals. The minimum sample size was largely influenced by the studied spine and fiber traits, ranging from 1 to 70 spines. The results provide pioneer information useful in spine sample collection in the field for taxonomical, biomechanical and structural studies. Nevertheless, similar studies should be carried out for other cacti species to make generalizations. The large spine and fiber variation within areoles observed in our study indicates a very complex spine morphogenesis.Although spine variation within cacti species or populations is assumed to be large, the minimum sample size of different spine anatomical and morphological traits required for species description is less studied. There are studies where only 2 spines were used for taxonomical comparison amnog species. Therefore, the spine structure variation within areoles and individuals of one population of  Cactaceae family. Cactus spines are the modified bud scales of an axillary bud, originating from primordia which are morphologically indistinguishable from the leaf primordia  Slaba population with the intent to solve three questions: (1) does areole position on the plant body play an important role in spine and fiber variation?; (2) are spine and fiber traits less variable within an PageBreakareole than between areoles and individuals?; (3) how many spines need to be collected for an analysis of specific traits? Our results will provide useful information for spine sample collection in the field for taxonomical, biomechanical, physiological or structural studies.Spine and fiber variation within areoles and individuals was studied in one Gymnocalycium, Gymnocalyciumkieslingiisubsp.castaneum was chosen for the study. This is an endemic taxon of the Argentinian province of La Rioja, which is taxonomically clear with relatively low morphological variability. It grows in 13 populations occupying fairly narrow ecological niche. It grows on the poor and highly permeable sandy soils without humus, blended with skeleton basement rock, generated by the disintegration of granitoids on the slopes of the Sierra de Velasco Mts. The climate is semi-arid and the mean annual precipitation is 360 mm. The mean annual temperature is 20 °C with 2,800 hours of sunshine . Plants are uniformly found under/or outsides of Larreacuneifolia Cav. in the phytogeographical district Monte (2) compared to other species of the subgenus. It lies between city of Aimogasta in the north and city of Villa Sanagasta in the south.A single representative of the nominate subgenus of ct Monte  at altit67°07’S; 29°22’W). Fifteen plants were randomly selected on a ca. 700 m line along the road (north-south), at distances of 50 m from each other. From each plant, one areole from the basal, middle and upper part on the northern face of the plant body were sampled . Additional areoles were sampled from 5 plants at each examined position for scanning electron microscopy (i.e. 15 areoles). Only fully developed areoles were collected.The plant usually forms flattened spherical bodies 60–90 mm in diameter. It rarely achieves a greater height than width . The plaSEM using a VEGA TS 5130 instrument  operating at 15 kV. Images of the whole areole and detailed images of the spine surface in the middle and top spine parts were made. From these images, epidermis characteristics, shape of epidermis cells, presence and type of trichomes and presence of fissures were determined.The whole areole was mounted on specimen stubs, sputter-coated with gold, and observed with high-vacuum PageBreakl) was measured before making cross-sections. Handmade cross-sections were taken from the spine base and were examined under a bright field microscope  at magnifications up to 400× and were photographed using a digital camera  connected to a computer with the QuickPhotomicro 2.3 software . Spine width (Sw), spine thickness (Sth), spine cross-section area (Sa), spine circumference (Sc), fiber maximum and minimum diameter (Fmax and Fmin), and cell wall thickness (CWth) were measured using the ImageTool 3.00 software   were defined as the Sw/Sth and Fmax/Fmin ratio, respectively, with a ratio of 1 denoting a perfectly round cross-section and larger ratios indicating a more ellipsoidal shape. In total, 245 spines were examined.A 5% solution of hydrochloric acid was used to soften spines before sectioning. In this solution, two days of soaking was sufficient to soften the spines for anatomical analysis. The spine length (Slinear mixed effect models (LME). In these models, all of the traits come from a nested design; therefore, we used LME to avoid the problem of pseudoreplication . In the LME analyses, traits were treated as factors with fixed effects, and individuals and areoles were treated as factors with random effects. LMEs were fitted using the LME function in the NLME library of the R statistical program )^{2}}{D^{2}}\]\end{document}standard deviation (SD) for the group, the (t1-α/2(n-1)) value is the quantile of the Student’s t-distribution for n-1 degrees of freedom and D is the desired margin of error. The interval limits for minimum sample size were taken as 10, 15 and 20% differences of the mean. Only spines sampled from the middle part of plant body were used for minimum sample size calculation. Calculations were performed in the R software environment  ranged from 3 to 16 mm and spine cross-section area (Sa) from 0.21 to 2.81 mm2 (Table l and Sa had the largest coefficient of variation (CV) between individuals (Table r) had the lowest CV (Table r was 1.25 (Table max) and fiber minimum diameter (Fmin) were 13.7 and 10.03 µm, respectively (Table r) was the least variable PageBreaktrait  had the highest CV of the fiber traits  (Table The fiber traits were less variable than spine traits between areoles Table . Althougr) Table . In gener) Table .CV of spine traits within an areole was lower than between areoles and individuals  and for Sl (60% of sampled areoles)  and for Fmax (44% of sampled areoles). On the other hand, a CV of CWth higher than 15% was only found for 7% of the sampled areoles.The ls Table . NeverthSpines within an areole were distributed only marginally with radial arrangement Fig. . All spia or Sc, 25 spines would be needed and even fewer spines would be needed for Sr  of Mammillariascrippsianavar.armeria and Echinopsis sp. have a smooth surface, whereas older spines (situated in the lower parts of plant body) show a broken surface. We did not observe such differences, which may be specific characteristics of a particular genus of cacti. However, there were differences in spine surface damage and visibility. The spine surface was more visible and less damaged in spines from the upper part than in the lower parts. In the basal part, the spine surface was almost completely covered with mineral deposition . In the present study, areoles with 3 and 4 spines were also found in 11% from all areoles. The spines shape was the least variable trait and it was mostly circular. This is in accordance with l and Sa were the most variable spine traits. In another study, spine length was also found to be the most variable trait in 30 Aylostera and Rebutia (Cactoideae) hybrids  and vary considerably across species in number, length, width and thickness . This is hybrids . It has  hybrids . For exarainfall . It is orainfall . Howeverrainfall . Thus, t unknown  and the Gymnocalyciumkieslingiisubsp.castaneum were mostly oval and had a pillar structure. In our study, the mean fiber maximum and minimum diameters were in the range reported for Echinocactusgrusonii (Opuntiaficus-indica (Turbinicarpus sp. div. (th). Turbinicarpus species had a mean CWth of 0.7 to 5.9 µm  were described by grusonii , Opuntias-indica  and Turbsp. div. , which ho 5.9 µm , and Esc3–4.0 µm . These vCV for different studied traits within an areole was still high. For example, the CV for Sa was even higher than 40% in a few cases  had different values in the lower parts than in the upper part of the plant body. Nevertheless, the growth of cacti is very slow, and determination of their age without direct observation is difficult , Fr (6 spines), Sa (23 spines) and Sc (25 spines). In contrast, more spines should be collected to study CWth (70 spines), Sl (52 spines), Fmax (43 spines) and Sw (40 spines). These sample sizes may, however, only apply to spines collected from different plants within a single population. We should note that more similar studies on other cactus species or on the same species, but growing in different environment conditions are needed to make generalizations for spine sample collection in the field. This is important task for the correct delimitation and identification of cactus species especially if cultivated specimens were used as sources of evidence in taxonomy . The spine and fiber variation within an areole was lower than between areoles, but the variation was still high (Question 2). The minimum sample size was largely influenced by the examined spine and fiber trait, ranging from 1 to 70 spines (mean ± 10%) (Question 3). The large spine and fiber variation between individuals and even within single areoles observed in this study indicates a very complex spine morphogenesis. We encourage a further research focus on the spine and fiber variation in other cacti species, but also on the factors controlling the basal meristem function and gene expression in spines.


Of these, 36 studies were conducted in healthy participants and 14 on cognitively impaired individuals (including Alzheimer’s and dementia). Vitamin C status was measured using food frequency questionnaires or plasma vitamin C. Cognition was assessed using a variety of tests, mostly the Mini-Mental-State-Examination (MMSE). In summary, studies demonstrated higher mean vitamin C concentrations in the cognitively intact groups of participants compared to cognitively impaired groups. No correlation between vitamin C concentrations and MMSE cognitive function was apparent in the cognitively impaired individuals. The MMSE was not suitable to detect a variance in cognition in the healthy group. Analysis of the studies that used a variety of cognitive assessments in the cognitively intact was beyond the scope of this review; however, qualitative assessment revealed a potential association between plasma vitamin C concentrations and cognition. Due to a number of limitations in these studies, further research is needed, utilizing plasma vitamin C concentrations and sensitive cognitive assessments that are suitable for cognitively intact adults.Vitamin C plays a role in neuronal differentiation, maturation, myelin formation and modulation of the cholinergic, catecholinergic, and glutaminergic systems. This review evaluates the link between vitamin C status and cognitive performance, in both cognitively intact and impaired individuals. We searched the PUBMED, SCOPUS, SciSearch and the Cochrane Library from 1980 to January 2017, finding 50 studies, with randomised controlled trials (RCTs,  Vitamin C is essential for the formation of procollagen which then acts as an intracellular “glue” that gives support, shape and bulk to blood vessels . Studies2O2 and is oxidised in the process to an ascorbate free radical and DHAA. Ascorbate also supports the regeneration of other antioxidants, such as vitamin E and glutathione [Neurons are especially sensitive to ascorbate deficiency, possibly due to 10-fold higher rates of oxidative metabolism than supporting glia . Ascorbatathione .Indicative of its vital role in the brain is its recycling, homeostatic mechanism  which maAlthough higher plasma ascorbic acid concentrations generally result in higher CSF concentrations, these concentrations start to reach a steady state. As plasma concentrations decline, relatively more ascorbate is pumped into the CSF in order to maintain homeostasis . Studiesm) for ascorbate appears to be somewhat high (113 µmol/L); this affinity corresponds well to plasma ascorbate concentrations of 30–60 μmol/L [Thus, not only is it difficult to deplete brain ascorbate, it is also difficult to increase levels above those set by uptake and recycling mechanisms. In neuronal cells, the apparent Michaelis–Menten transport kinetics . Appraisal was determined using established guidelines for randomised, controlled trials (RCT), and observational studies (prospective and cohort) established from the Cochrane collaboration . QualityAn initial survey of the literature revealed that many studies used comparable cognitive and vitamin C measures—The Mini Mental State Examination (MMSE) and blood plasma vitamin C concentrations. Given this consistency in measurement we decided to further explore these trends across studies. A brief summary of these inclusions and methods is presented below. We contacted authors for mean values and standard deviations of studies which did not report numerical mean vitamin C concentrations or MMSE scores (0–30) but instead placed the means into categories .Given the practicality and accuracy of measuring absorbed vitamin C status through blood plasma, plasma vitamin C has been considered the ideal measure of vitamin C status . A numbeThe MMSE is a simple validated and reliable paper and pen questionnaire designed to estimate the severity and progression of cognitive impairment and used to follow the course of cognitive changes in an individual over time . Any scot-tests were conducted, comparing the baseline blood vitamin C concentrations and baseline MMSE scores between cognitively intact and impaired participants. Due to the ordinal nature of MMSE scores and ratio scales for blood test concentrations, a Spearman’s correlation coefficient analysis  was conducted. R-squared values, assessing goodness of fit and test of normality were conducted to establish the correlation between mean vitamin C concentrations and MMSE scores.Using IBM SPSS  Only studies which measured blood vitamin C concentrations and cognition through the MMSE were compared. Comparable mean vitamin C blood concentrations and MMSE scores were extracted as separate data points from each of the studies and plotted graphically. A number of studies assessing cognitively impaired individuals also used healthy controls. The mean MMSE and vitamin C concentrations from these controls was added to the mean scores of other cognitively intact samples for comparison.FFQ-based vitamin C levels were also converted to predicted blood concentrations, where every 1.97 mg of consumed vitamin C equates to 1 µmol/L of ascorbate plasma. A constant plateau in ascorbic acid concentration (60–80 µmol/L) is reached at 150 mg of consumed vitamin C . Given tAdditionally, qualitative analyses were conducted on the studies that utilized a range of other cognitive assessments and direct plasma vitamin C measures. These studies were reported qualitatively due to a large diversity in cognitive assessments and statistical reporting of results . The overall trend of results and quality of these trials was taken into account for the qualitative analysis.The search captured exactly 500 articles, of which 50 studies were included in the systematic review . Of thesIn the cognitively impaired samples, eight out of 14 studies used blood tests to measure vitamin C ,46,47,48t-tests revealed that mean vitamin C concentrations in the cognitively intact subgroup were significantly higher than in the cognitively impaired (t (15) = 4.5, p < 0.01) and mean MMSE scores were also significantly higher in this subgroup (t (10.3) = 5.7, p < 0.01).Mean MMSE scores and measured or derived blood vitamin C concentrations are plotted in In the cognitively impaired subgroup, there was a wide distribution of both MMSE scores (mean score range = 1.9–26.9) and vitamin C concentrations (19–44 µmol/L) . Mean viIn the cognitively intact subgroup, mean vitamin C and MMSE scores were extracted from 5 studies . In this group, mean vitamin C concentrations (Mean score ± SD = 54.9 ± 16) µmol/L) were widely spread (33.7–80 µmol/L) but mean MMSE scores (Mean score = 28.1 ± 0.7) were not (27.2–28.9). The lack of variance in MMSE scores precluded correlational analysis in this subgroup.2 value of 0.0016 revealed low variance and a spread in means around the fitted regression line. The Spearman’s correlation also revealed no significant correlation between MMSE scores and vitamin C concentrations (rs (11) = 0.009, p = 0.98).In the cognitively impaired subgroup the scatterplot /Table 4 A number of studies ,50,78,81Additional studies using cognitively intact groups of participants  assessedThe predicted blood vitamin C concentrations generated from FFQs in the cognitively intact participants when plotted , were ren = 7, n = 391 participants). In contrast, correlation analysis between blood vitamin C concentrations and MMSE scores in the cognitively intact studies was not feasible due to the low variance in MMSE scores, demonstrating the unsuitability of the MMSE in the cognitively healthy participants. Quantitative assessment of those studies in the cognitively intact groups revealed a potential association between plasma vitamin C concentrations and cognition. Our findings are consistent with a number of studies [This review evaluated 50 studies exploring the link between vitamin C and cognitive function. Extrapolated mean vitamin C concentrations and MMSE scores from a number of these studies indicated that the cognitively intact groups of participants had higher mean vitamin C concentrations and MMSE scores than the cognitively impaired groups. However, there was no significant correlation between mean vitamin C concentrations and mean MMSE scores in the cognitively impaired studies . These issues arise as a result of a lack of full appreciation of the redox chemistry and biology of ascorbic acid . A numbeA majority of studies included in this review failed to thoroughly explain blood sample handling and biochemical analysis. Ideal handling conditions of samples intended for ascorbate analysis include immediate coverage from light, immediate plasma isolation, rapid acidification, and freezing below −20 °C to avoid misinterpretations compounded by the use of poorly preserved samples . In ordeUnderestimation of vitamin C concentrations could occur if samples were not handled properly. Frequent freeze-thaw cycles or exposure to any metals (such as iron in the haemolysis of red blood cells) could both lead to rapid degradation of vitamin C in the sample . It has Several limitations can arise from the use of FFQs in determining nutrient level . Plasma While converted FFQ-based vitamin C levels were of a similar range to blood concentrations, this conversion needs to be interpreted with caution. The conversion ratio of 1.95 mg to 1 µmol/L in plasma was based on a study that used 8 healthy participants . HoweverPlasma vitamin C differs according to polymorphisms of sodium dependent active transporters (SVCT2 and SVCT1) despite equivalent vitamin C intake indicating that SVCT1 and 2 genotype may determine the strength of the association between vitamin C intake and circulating vitamin C concentrations . Some peIn addition, dietary assessment has reliability and validity issues in relation to even mild cognitive deficits, which are frequent in older populations . These iMoreover, the reviewed randomised controlled studies have failed to assess the effects of a vitamin C intervention on its own, by using multivitamins. A large portion of the included studies have made efforts to statistically control for potential confounders. Although our review did demonstrate lower plasma vitamin C concentrations in the cognitively impaired, other studies using impaired samples have shown depletions in a number of other vitamin and minerals including: vitamin B12 , vitaminMoreover, it can be speculated that a consistently high Vitamin C status acts in a preventive manner, while vitamin C supplementation per se is not a treatment for clinical AD . Thus, iIntake at the time of measurement may not reflect lifetime dietary habits and given data that suggest that amyloid plaque burden begins to form well before middle age , intakesIn addition to the limitations on vitamin C levels, there were limitations regarding the type of cognitive measures. A number of long term prospective studies incorporated cognitive tests suitable for screening and assessing the incidence of Alzheimer’s, such as the MMSE. Given the simplicity of such tests, and the scales used to measure performance, it becomes difficult to establish cognitive changes unless the cognitive decline is extremely severe. These MMSE scales have been effective in measuring cognition in those clinically diagnosed with a neurodegenerative condition ,48, and The sensitivity of the MMSE to detect differences in cognitively intact samples has been questioned ,123. ThiA further limitation to be considered is the often self-selection of healthier, more cognitively-able population in population studies. As a consequence of high baseline performance in cognitively intact participants, ceiling effects with narrow ranges in results can occur . This efIn cognitively intact samples, cognitive tests sensitive to age-associated cognitive decline should be employed to maximize the observation of any potential effects. Programs such as The Cambridge Neuropsychological Test Automated Battery  and The Future studies should incorporate a number of recommendations. Firstly, the most reliable and practical measure of vitamin C is the measurement of biological blood samples. Moreover, the incorporation of FFQs would allow a measure of possible confounding variables . Age-sensitive cognitive tests assessing response time and accuracy should be administered , particuIn summary, studies included in this systematic review demonstrated higher mean vitamin C concentrations in the cognitively intact groups of participants compared to the impaired groups. No correlation was found between vitamin C concentrations and MMSE scores in the cognitively impaired groups of participants. Analysis of the studies that used a variety of cognitive assessments was beyond the scope of this review, however, qualitative assessment in the cognitively intact groups revealed a potential association between plasma vitamin C concentrations and cognition. Due to a number of limitations, further research, assessing plasma vitamin C concentrations, taking confounding factors such as vitamin B12 and vitamin E into account, and the use of more sensitive cognitive assessment methodology for cognitively intact participants are needed to provide more insights into the relationship between vitamin C and cognition.


Cephalorhynchus eutropia, has a continuous coastal distribution with strong genetic differentiation identified by nuclear DNA markers. We explored the historical dimension of this genetic differentiation between northern and southern populations to evaluate phylogeographic structure. Additionally, we conducted mtDNA and microsatellite analyses to detect past and recent demographic changes. The southern population was characterized by lower genetic diversity with a signal of population expansion, likely associated with ice retreat and habitat extension after the Last Glacial Maximum (LGM). In contrast, structure within the northern population was more consistent with stable historical population size. Approximate Bayesian Computation analyses suggested that during the LGM, C. eutropia persisted in the northern area; while the south was colonized by dispersal ~11,000 years ago followed by population expansion. This study shows that Chilean dolphin population structure is consistent with predictions from the Expansion-Contraction biogeographic model, with a poleward post-glacial shift revealed in current genetic structure. The results also confirm the validity of the population units previously identified, demonstrating their historical origin and highlighting the utility of integrating genetic markers with different temporal scale resolutions.The complementarity of historical and contemporary processes contributes to understanding the genetic structure of continuously distributed marine species with high dispersal capabilities.  Thunnus thynnus thynnusCarcharodon carcharias, in the Mediterranean SeaMegaptera novaeangliae, in the North PacificUnderstanding the population structure of marine species represents a challenge as their environments exhibit fewer obvious physical geographic barriers to gene flow than terrestrial onesa priori hypotheses regarding the placement of subpopulation boundaries or patterns of genetic structure may help to explain the observed patterns712Eleginops maclovinusGalaxias maculatusGenetic structure in marine populations commonly reflects a historical and contemporary interplay among a combination of ecological, demographic, behavioral, genetic, oceanographic, climatic and other factorsDelphinapterus leucas, in the western Nearctic occurred after the retreat of the Pleistocene ice cover, and the chronology of postglacial events is thought to have shaped the dispersal routes of this species from its glacial refugesLagenorhynchus albirostris) in the North Atlantic, demographic inference based on mitochondrial DNA suggests a population expansion in the Barents Sea off the Norwegian coast and geographic extension to the British Isles in the North Sea after the Pleistocene glacial eventsPhocoena phocoena, and minke whale, Balaenoptera acutorostrata, in the North Sea were mainly attributed to environmental requirements for feeding and breedingEschrichtius robustus, in the North Pacific were also modified by variations in sea level and the availability of environments not covered by ice in the late PleistoceneClimatic and oceanographic variations have also contributed substantially to the current distribution pattern, geographic variation and population structure of some species of the Order Cetacea. The population expansion of belugas, Cephalorhynchus eutropia), distributed along the Southeast Pacific coast between 30° and 56°S, represents an interesting model for understanding how a combination of historical and contemporary factors resulted in the current genetic structure of this continuously distributed marine species. Using 21 microsatellite markers, Pérez-Alvarez et al.ST = 0.15 and RST = 0.19) were higher than those reported among populations of most other dolphin species at largeThe coastal endemic Chilean dolphin  the northern area is located from 30°S to 42°S  characterized by an open and exposed coast with the presence of river runoff, where Chilean dolphins are associated with estuarine habitats29C. eutropia may have survived the Last Glacial Maximum (LGM) in low latitude areas, contracting its distribution to areas less impacted by ice, and then recolonizing higher latitudes through range expansion after the ice retreatC. eutropia to test whether population structure as influenced by the LGM is more consistent with the predictions of the expansion-contraction model under the “In situ refugia” or “Northern shift” scenarios.Consistent with the basic E-C model, Analyzing the microsatellite and mtDNA data simultaneously, the spatial model in GENELAND identified two genetic clusters with a boundary located between Maullín (41°36′S) and Aysén (46.36′S)  and 2. IST = 0.39, P < 0.0001 and FST = 0.19, P < 0.0001). Phylogeographic structure was indicated by the significantly greater value for ΦST compared to FST (P = 0.0004).A total of 14 haplotypes were defined by 15 polymorphic sites among the 64 individuals. Overall, mtDNA haplotype diversity (h) was 0.75 ± 0.053 and nucleotide diversity (π) was 0.038 ± 0.002 . The SouAnalysis of Bayesian skyline plots of each genetic group showed that the Northern population of Chilean dolphin  remainedC. eutropia along its distribution. The results supported the “Northern shift” scenario with the highest posterior probability 0.81 (CI 95% 0.801–0.825) and 0.99 (CI 95% 0.926–1.000) for mtDNA and microsatellites respectively. Additionally, model checking, direct approach and logistic regression also supported the Northern shift scenario to be the most likely (The ABC (Approximate Bayesian Computation) analysis discriminated between the two proposed scenarios based on geographic population structure and demographic inference of t likely . This suLeopardus guigna in southern South America, where geographical barriers such as the presence of the Andes Mountains and the Chacao Channel have shaped its current genetic population structureOrestias ascotanis, endemic to the Altiplano, where population structure reflects a long-term consequence of habitat fragmentationThe interpretation of the structure and distribution patterns of genetic diversity is one of the research objectives of fields including molecular ecology, population genetics and landscape geneticse.g. using microsatellite locie.g. using mtDNAHowever, as few absolute barriers to gene flow exist in oceans, even widely separated regions may be genetically connected and many marine species often exhibit low levels of genetic differentiation over large distances34Cephalorhynchus is an example of marine species with widespread but discontinuous distribution in the cool-temperate zone of the Southern HemisphereC. commersonii; Hector’s dolphin, C. hectori; and Chilean dolphin, C. eutropia) have demonstrated genetically divergent and structured populations. In the Commerson’s dolphin, genetic differentiation has been described for populations found along the southeastern coast of South America and around the Kerguelen Islands in the southern Indian Ocean  and the absence of a geographic gap or barrier separating the genetically distinct populations make its current genetic structure puzzling. Nevertheless, from a historical point of view, the genetic break between Northern and Southern populations appears to coincide with the boundary between two major marine biogeographic units31vs. non-affected areas during glacial cycles.The dolphin genus in situ refugiaST = 0.15 and RST = 0.19, Pérez-Alvarez et al.ST = 0.39, P < 0.0001 and FST = 0.19, P < 0.0001). The geographical boundary between these two population units coincides with the extension of the LGM ice sheetA signal of population structure is predicted, as recolonization from non-affected areas will likely produce genetic differentiation between areas affected and others not affected by the glacial cycles due to genetic drift or alternatively, long-term isolation of populations that persisted within Less genetic diversity is predicted in areas recolonized postglacially compared to populations that remained in refugia or at lower latitudes as consequences of the ice-free recolonization process, founder effect and genetic driftFinally, population growth that follows the recolonization process is predicted to generate a population expansion signal in deglaciated areas. For the Chilean dolphin, Northern and Southern populations exhibit different demographic histories. Demographic inference analyses , revealed a relatively stable population size for the last 40,000 years for the Northern population, after which a non-significant trend of population decrease is observed. This signal could reflect a current decrease in this population maybe related to human activities. In contrast, for the Southern population, Bayesian skyline plots as well as ABC estimated parameters, showed a population size increase from approximately 11,000 years ago. In both cases, coalescent method  tends toIn this context, the present study shows that the predictions of the Expansion-Contraction model are consistent with the population structure, genetic diversity and demographic history of the Chilean dolphin:Overall, our results suggest that the southern Chilean dolphin population went through an expansion process after LGM ice retreat by population expansion initiated about 11,000 years ago. This is consistent with the chronological dating information of glacial cycles reported for these latitudes, particularly during the LGM. The time period estimated for the glacial advance to the northern limit of Pacific Patagonia is approximately 17,900 years ago, and the deglaciation would have begun about 17,500 years ago, synchronously throughout Patagonia35in situ refugia” scenario involves the persistence of species in refugia within the glaciated area, followed by expansion within the higher-latitude area from those refugia. The “Northern shift” scenario involves a contraction in population size and displacement toward lower latitudes during periods of cooling, followed by recolonization of southern glaciated zones during postglacial warming from the northern area. Based on geographic population structure and demographic inferences, The Approximate Bayesian Computation approach supported the “Northern shift” as the more probable scenario. This scenario essentially amounts to restriction of the distribution of C. eutropia during the LGM to the area north of Chacao Channel. The exclusion of C. eutropia from the southern area during the LGM may be explained by decreased primary productivity, prey distribution and abundance decrease within the area affected by the LGM, and simple physical displacement from ice-covered areas. The absence of this species in areas directly affected by the glacial cycles is consistent with what is known about genus Cephalorhynchus, which appears to be restricted to particular and limited habitats in the cool-temperate zoneThe population genetic diversity and structure of the Chilean dolphin can be analyzed relative to two alternative E-C historical scenarios. The “Lontra provocax)i.e. “in situ refugia”) as the most plausible scenario for that species during the LGM. This is mainly based on the absence of a star-shaped haplotype genealogy, no sign of population growth for the area, and high levels of genetic diversity observed in the area affected by the ice. The persistence of the southern river otter in the glaciated area would have been possible given the survival of other aquatic and marine species that serve as its food source and/or flexibility in its diet as an adaptation to the changing environment during the LGMIn contrast to the findings for the Chilean dolphin, a study on another aquatic mammal found in similar latitudes, the southern river otter  was amplified for 64 individuals using the primers M13 Dlp1.5 5′-TGTAAAACGACAGCCAGTTCACCCAAAGCTGRARTTCTA-3′ and 8G 5′GGAGTACTATGTCCTGTAACCASequences were edited and aligned in PROSEQ 2.91 and the species was confirmed using a BLAST search undertaken in GeneBank and by DNA Surveillance.et al.C. eutropia samples and 21 dinucleotide microsatellite loci: Ev1, EV14, EV37, EV94, EV104We used genotype data obtained by Pérez-Alvarez, To explore spatial boundaries we used GENELAND, a Bayesian model that uses genotypes and spatial coordinates of individuals to cluster them into populations at approximately Hardy-Weinberg equilibrium, considering linkage equilibrium between loci. For this analysis, both markers (mtDNA and 21 microsatellites loci) were used simultaneously. An allele frequency uncorrelated model was set, with 1,000,000 MCMC iterations and thinning of 100.ΦST  and FST (using haplotype frequencies) in ARLEQUIN v 3.5. Phylogeographic structure was evaluated using PERMUT Software by comparisons between the observed ΦST and FST values, using 10,000 permutations. Because FST is a genetic differentiation index between populations based on haplotype frequency and ΦST considers both haplotype frequencies and genetic distance between haplotypes, a higher value of ΦST compared to FST reveals the existence of a phylogeographic structure.To evaluate the existence of phylogeographic structure, control region sequences were divided into two groups based on results obtained from GENELAND. Genetic diversity at the haplotype (h) and nucleotide (π) levels was estimated for the complete study area and also for the Northern and Southern areas using ARLEQUIN v 3.5. Considering the differences in sample size between areas, values of haplotype richness were also adjusted to sample size using a rarefaction analysis in PAST software. Genealogical relationships of the haplotypes were investigated by constructing a median-joining network in NETWORK 4.5.1.0. Genetic differentiation between Northern and Southern populations was assessed by calculating To assess the patterns of demographic history (1) The past population dynamics (demographic history) of each population was reconstructed by a Bayesian Skyline-plot method implemented in BEAST 1.7, which is based on coalescent theory and quantifies the relationship between the genealogy of the sequences and the demographic history of the populationC. eutropia, we used the Approximate Bayesian Computation (ABC) method. This approach makes it possible to test complex population genetic models by simulating the data sets of predefined scenarios and comparing their summary statistics with the summary statistics of the observed datain situ refugia” scenario proposed that the population of C. eutropia present in the southern area of distribution (Chacao Channel to the South) experienced reduced population size and survival only in restricted areas during the Last Glacial Maximum (LGM). Subsequently, a population expansion would have occurred in this area after the retreat of the ice. The “Northern shift” scenario considered that during the LGM the distribution of C. eutropia was restricted to the area north of Chacao Channel. In this case, the Southern population was originated after the ice retreat through postglacial colonization from the Northern population, and was characterized by founder effects followed by rapid population expansion .


Late-onset Pompe disease (LOPD) is a lysosomal storage disease resulted from deficiency of the enzyme acid α-glucosidase. Patients usually develop a limb-girdle pattern of myopathy and respiratory impairment, and enzyme replacement therapy (ERT) is the only specific treatment available. Recently, LOPD has been associated with low bone mineral density (BMD), but the effect of ERT on BMD is inconclusive. In this report we described our early observations on the change of BMD after ERT in Chinese LOPD patients.We studied four Chinese LOPD patients with different severities of myopathy. All were underweight, and three had osteoporosis at baseline. We found significant weight gain in three patients after ERT and all four patients showed improvement in BMD. The biggest improvement, 84.4% increase in BMD, was seen in a lady with the most prominent weight recovery. Our results suggest that ERT improves BMD in Chinese LOPD and weight gain could be a major contributor to this effect. Pompe disease is an autosomal recessive disorder caused by the deficiency of a lysosomal enzyme, acid α-glucosidase, resulting in glycogen accumulations and consequent autophagic buildup –3. The lRecently, low bone mineral density (BMD) and osteoporosis has been reported in LOPD , 7. Thesl-alanine supplements to all of our LOPD patients. Patient 3 and patient 4 also received alendronate for osteoporosis after the baseline DXA study. Alglucosidase alfa infusion was given at the standard regime of 20 mg/kg every 2 weeks.Four Chinese LOPD patients who underwent DXA  at baseline before ERT and a follow-up study after ERT were included. Patients 1 and 2 were brothers, patients 3 and patient 4 were sisters. Patient 1 was a juvenile-onset patient with an aggressive disease course; he was wheelchair-bound and required full-day non-invasive ventilation (NIV) support upon initiation of ERT. Because of his previous spinal surgery for scoliosis, patient 1’s DXA was performed on the forearm and hip instead of the usual lumbar spine and hip. Patient 2 was fully ambulatory and did not require NIV. Both patient 3 and patient 4 presented with type II respiratory failure in their early 30s. They were ambulatory and on nocturnal NIV. We prescribed calcium, vitamin D and We serially monitored the patients’ mobility and pulmonary function with the 6-min walk test (6MWT) and spirometry, respectively, according to our internal management protocol for all LOPD patients on ERT, in order to justify the continuation of ERT in these patients through public funding. We also used a modified Walton scale according to Slonim et al.  to assess the muscle weakness in relation to their daily functions . A folloThe four patients were in different stages of severity across the disease spectrum Table . They weIn the follow-up reassessment after ERT Figs. ; Table 2Significant weight gains of 22, 21 and 27% was observed in patients 1, 2 and 4, respectively. Patient 3 did not gain weight after ERT and remained severely underweight. BMD had increased in all four patients after ERT. There was an increase of approximately 10% in patients 1, 2 and 3 and an astonishing 84.4% at the hip in patient 4. The improved Z-scores showed that patients 1, 3 and 4 had moved from osteoporosis to osteopenia after ERT.Low BMD causing a predisposition to fracture has been associated with LOPD , 12, butOur four patients represented a spectrum of different disease severities, but they were all underweight at baseline before ERT. Three had osteoporosis, and one had normal BMD. All four patients showed improvement in BMD in the follow-up study. Patient 4 had the most prominent weigh gain, and her physical endurance improved, but there was no significant enhancement in her hip girdle muscle strength, and her mobility was only modestly better. Her huge gain in BMD was more likely a result of her recovery of body weight. Alendronate could not improve BMD by that much, and any positive effect from improvement in muscle strength should be modest. Patients 1 and 2 were both underweight and had similar weight gain after ERT. Patient 1 remained severely disabled, and patient 2 had good improvement in physical performance. The improvement in BMD was more marked in patient 1 than in patient 2, suggesting that the treatment effect could be more obvious in those with a lower baseline BMD and that strength might be less important than body weight. These observations agreed with the previous reports that described inconsistent ERT responses in LOPD patients with diverse characteristics , 18. NevAlthough it is acknowledged that Chinese LOPD patients commonly manifest an aggressive clinical course and low BMI is a poor prognostic marker in LOPD, the reason for such a high prevalence of malnutrition in Chinese LOPD is still poorly understood. Postulations include, among many, a chronic malabsorption state from gastrointestinal smooth muscle involvement in LOPD and a persistent catabolic state from respiratory failure , 20. HowWe observed a positive effect on BMD after ERT in a small group of Chinese LOPD patients, and suggested a few postulations to explain this association. However, our findings were mainly hypothesis generating, we were unable establish any causal relationship between ERT and BMD. Future studies should focus on catabolism, changes in body compositions, changes in bone turnover markers, and the balance of hormones and cytokines, both at baseline and with ERT, and take the secondary contributing factors into account to obtain a clearer picture on LOPD and bone health.


Background: Activated protein C (APC) inactivates factor V (FV) by cleavage of its heavy chain at Arg306, Arg506, Arg679, and Lys994. Mutational changes, which abolish APC cleavage sites, may predispose thrombosis by altering the inactivation process of FV. FV Leiden (FVL) (Arg506Glu) has been demonstrated as a strong risk factor for thrombosis. In the current study, we have studied whether mutations in the cleavage sites of FV for APC, not due to FVL, would have a role in presenting APC resistance (APCR) and initiation of a cerebral thrombotic event.Methods: A group of 22 patients with a history of cerebral venous thrombosis (CVT), who were not carriers of FVL enrolled in the study. The patients who had conditions associated with acquired APCR were excluded from the study. APCR test was performed on the remaining 16 patients, which showed APCR in 4 plasma samples. DNA sequencing was performed on four exons of FV of APCR patients, encoding Arg306, Arg506, Arg679, and Lys994.Results: Mutations were not found within nucleotides encoding the cleavage sites; neither was found within their close upstream and downstream sequences.Conclusion: Our results show that polymorphisms affecting cleavage sites of FV other than Arg506Glu it would be less likely to be the basis for APCR and its increased thrombosis susceptibility. In addition, it emphasizes on the importance of screening for APCR in the patients diagnosed with CVT. Furthermore, the risk for developing a CVT is higher in patients with hypercoagulable states such as hyperhomocysteinemia, protein C, protein S and antithrombin III deficiencies, as well as careers of prothrombin gene and factor V Leiden (FVL) mutation.1,2 Accordingly, the prothrombotic screening for hypercoagulable states due to genetic and acquired disorders is an important part of investigating the etiology of CVT.1,2Cerebral venous thrombosis (CVT) is an uncommon but serious type of cerebral vascular events which mostly affects young adults and children.3 APC can down regulate FV procoagulant activity by cleavage at Arg306, Arg506, Arg679, and Lys994.3,4 Mutational changes which abolish APC cleavage sites may dilute its effect on inactivation of FV. FVL, which is the result of a single point mutation at Arg506, is a common and strong hereditary risk factor for thrombosis in Caucasians.5,6 The APC resistant (APCR) phenotype which is associated with a 7-fold increase in the risk of deep vein thrombosis, is considered to be a consequence of FVL in 95% of cases.5Activated protein C (APC) plays a key role in the anticoagulant pathway by inactivating FVa and Factor VIIIa. There are several proteolytic cleavage sites on FV that is responsible for activation and inactivation of the molecule.7,8Acquired conditions including cancer, pregnancy, using oral contraceptive pills (OCP) and hormone replacement therapy (HRT), elevated Factor VIII levels ≥ 150 IU/dl and positive lupus anticoagulant results may also be the cause of APCR.9 However, DNA-analysis has shown that some cases with APCR do not carry an R506Q mutation.10 Therefore, in these individuals, the inactivation of FVa by APC may be influenced by polymorphisms other than Leiden mutation. In the current study, we have studied whether mutations in the cleavage sites of FV for APC, not due to FVL, would have a role in presenting APCR and initiation of a cerebral thrombotic event.The functional assays that are being used for screening of APCR are widely considered equal to FVL screening test, especially in populations with high prevalence of FVL.11 22 patients accepted to take part in the current study performed at the Neurology Clinic of Alzahra University Hospital . Of each patient, a 5 cc sample of venous bloodstream was collected into a citrated tube. Poor platelet plasma was extracted after centrifuging and stored at −20 °C. Another 5 cc sample was collected in ethylenediamine tetraacetic acid tube and stored at −20 °C until molecular lab assessments.A group of 38 ≤ 65 year-old adults with a past diagnosis of CVT was recalled for this study. These patients had previously been checked for the presence of FVL using restriction fragment length polymorphism (RFLP) method, and the results were negative.Factor VIII, lupus anticoagulant and APCR tests were performed on 22 plasma samples of patients. To eliminate the conditions that could induce APCR without a Leiden mutation, the patients who had ≥ 150 IU/dl of Factor VIII or positive lupus anticoagulant test results were excluded from the study. None of them were pregnant, OCP consumers or under HRT at the time of sampling.Using HEMOCLOT Factor V-L kit , APCR was measured in the plasma samples of the patients. The ratio of clotting time was calculated in the presence and absence of APC. If this ratio was ≥ 2, the plasma is considered normal. In patients with significant APCR such as R506Q mutation, this ratio is lowered to ≤ 1.80. The ratio > 1.80 and < 2 is considered as borderline.DNA was extracted from blood samples of 4 APCR patients with a standard DNA isolation kit . Four primers were used for running PCR on the cleavage site regions: 12 A 267 bp fragment of exon 10, encoding Arg506 cleavage site, was amplified by following primer: Forward: 5'-TGCCCAGTGCTTAACAAGACCA-3’ and reverse: 5'-TGTTATCACACTGGTGCTAA-3’.5 A 226 bp fragment of exon 13, encoding Arg679 cleavage site, was amplified by the following primer: Forward: 5’-TGTCTGTCTCTCTTCTGTAAGAACT-3’ and reverse: 5’-CTGGTAATCATAGTCAGCATCAC-3’.12 The last primer: Forward: 5’-CAGCCCCCAGAATGCCTCA-3’ and reverse: 5’- AGACCTGGAGGACAGCTTGC-3’ was designed to synthesize a 432 bp fragment of exon 13, that encodes Lys994 cleavage site.A 228 bp fragment of exon 7, encoding Arg306 cleavage site, was amplified by the following primer: Forward: 5’-CTTGAACCTTTGCCCAGTGG-3’ and reverse: 5’-TTGTCTTTCTGTCCTAACTCAGC-3’.Polymerase chain reaction products were collected into sterile 500 µl tubes and stored in −20 °C until sequencing procedures . The results were compared with sequence information of FV in the National Centre for Biotechnology Information (NCBI) gene database.A total of 22 patients with a history of CVT, who were negative for FVL, were studied. There were 16 females (72.7%) and 6 males (27.3%) with a mean age of 36.77 ± 13.35 years. Three patients had a positive lupus anticoagulant test and were excluded from the study. The level of Factor VIII was increased in 4 patients, and they were excluded too (one patient excluded for both). Four patients out of remaining 16 patients (25%) showed resistance to APC in the absence of other acquired conditions by showing a clotting time ratio under 2. Two of them represented APCR by 1.4 and 1.6 clotting time ratio. The other two patients were in the borderline range (ratio 1.9).DNA sequencing was done on four regions of FV that encode Arg306, Arg506, Arg679, and Lys994 cleavage sites in four APCR patients. Mutations were not found within nucleotides encoding the cleavage sites; neither was found within their close upstream and downstream sequences.In this series of patients, the FV-mediated APCR was investigated in a selected group of CVT patients with no evidence for acquired causes of this thrombogenic condition. The sequencing study of four known cleavage sites of FV for APC, as potential sites for generating APCR, failed to find any polymorphism. Similar to the results of the previous testing with RFLP method, DNA sequencing on Arg506 confirmed the absence of FVL mutation. Thus, the APCR could not be explained by a molecular mechanism affecting the APC cleavage sites other than R506Q.13,14 It has been reported that 10-25% of CVT patients are carriers of this mutation.14,15 Among Iranian population, Rahimi, et al.16 found a significant correlation between FVL mutation and CVT in Iranian patients with Kurdish ethnic background. In contrast a study by Ashjazadeh, et al.17 indicated that frequency of FVL mutation were not significantly increased in CVT patients who lived in southern Iran with predominant Fars ethnicity.Mutated FVL which decreases the anticoagulant activity of APC by altering the main cleavage site of APC on FV, has been proposed to be strongly associated with CVT.11 As APCR was observed in some of our patients in the absence of FVL, looking for other genetic polymorphisms appeared to be the most logical approach. Mostly the polymorphisms that potentially act in a similar way as FVL does.Similarly, we observed a frequency of 5% in a group of 40 CVT patients and could not find a significant difference compared with controls.Our exclusion criteria resulted in a small group of 4 patients to be investigated out of 22 recalled CVT patients without FVL, but simultaneously, it would raise the possibility that APCR was a result of a genetic defect.4,18 The latter two cleavage sites are associated with cofactor inactivation in which cleavage at Arg306 is responsible for 70% loss of cofactor activity and the subsequent cleavage at Arg679 that occurs at a slower rate than that of Arg306 and Arg506, is responsible for the remaining 30%.18 Although there is a lack of studies on kinetics and clinical significance of APC-mediated cleavage at Lys994 in the literature, it appears to be a location of minor importance on cofactor activity of the molecule.19The cleavage of FV by APC at the cleavage site Arg506 is the most important part in the inactivation of FVa and results in a partial inactivation. It is also essential for optimal exposure of cleavage sites at Arg306 and Arg679 and subsequent inactivation of FVa.20 two reported polymorphisms at Arg306 have shown a milder APCR phenotype. Arg306→Thr (FV Cambridge) makes a mild APCR and Arg306→Gly (FV Hong Kong) appeared to have no change in the susceptibility to APC cleavage.21 Mutations at Arg306 are probably associated with low thrombotic risk.21,22Although the mutation at position Arg506 leads to a significant APCR and associated with 3- to 7-fold increase in the risk of thrombosis as heterozygous and up to 80 folds as homozygous,23,24 Hence, the APC cleavage is occurred very slower in these sites and to our knowledge have no physiologic significance in normal situation.23,24 They may have a role in the presence of mutant factors , but still remains to be studied.Theoretically, the mild APCR observed in our patients can be attributed as a result of other unknown cleavage sites on FV. The recent studies have demonstrated some novel cleavage sites close to Arg306 and Arg506.25 is a mutated variant that carries aIle359Thr substitution which provides an additional glycosylation at Asn357 that results in impaired inactivation of FVa. In addition, it appears to have a poor APC cofactor activity.25,26 The FV R2 haplotype, which is a collection of mutations in FV, also may represent a mild APCR in homozygous carriers.27The observed APCR could be attributed to other variants of v that display APCR phenotype without interfering its cleavage sites on FV. FV Liverpool28 our results stress the importance of APCR screening in patients with CVT.Our study could not find any genetic polymorphism of FV, at its multiple cleavage sites for APC. It shows that polymorphisms affecting cleavage sites of FV other than Arg506Glu it would be less likely to be the basis for APCR and its increased thrombosis susceptibility. In addition, as FVL and APCR are considered as independent risk factors for thrombosis,