Leishmania donovani and transmitted by Phlebotomus orientalis is endemic in various remote areas of north and north-west Ethiopia. The present study was designed to determine the sand fly fauna and bionomics of P. orientalis in the VL endemic focus of Tahtay Adiyabo district.Visceral leishmaniasis (VL) is a neglected tropical disease, which is strongly associated with poverty. VL caused by Sand flies were collected using CDC light traps (n = 602), sticky traps  and indoor pyrethrum spray catches (n = 578 house visits) from indoor, peri-domestic and agricultural field habitats between May 2011 to April 2012. All sand fly specimens collected were identified to species level and counted.Phlebotomus and sixteen Sergentomyia) were collected and identified. S. africana and P. orientalis made up 59.1% and 23.5% of the collected sand flies, respectively. As it could be determined from the proportion of collections from outdoor  and indoor locations, P. orientalis appears to exhibit increased exophilic behavior. The outdoor to indoor index was 79:1 on m2 of sticky traps. Mean density of P. orientalis caught was significantly higher on horizontally placed sticky traps (mean = 60 ± 14.56/m2/night) than vertically deployed sticky traps (12 ± 3.57/m2/night). The highest abundance of P. orientalis occurred between March and April. Through July to September, there was a sharp decline in abundance of P. orientalis population. Regarding climatic variables, P. orientalis density in light traps and on sticky traps showed a significant positive and negative association with temperature and relative humidity, respectively. However, non-significant negative correlation was observed with rainfall pattern.In total, 100,772 sand fly specimens, belonging to 25 sand fly species . Likewise, the people in the area usually sleep in compounds during these months that potentially expose them to a high risk of peri-domestic VL transmission.Overall,  Leishmania donovani, is an important public health problem in several regions of Ethiopia, with an estimated annual incidence of 3,700 to 7,400 cases per year  to fit normal distribution and checked for normality by Shapiro-Wilk test. When trapping data did not conform to the normal distribution, the non-parametric equivalent tests of Kruskal-Wallis and Mann–Whitney-erformed . For habPhlebotomus and sixteen Sergentomyia) were collected and identified (Table Phlebotomus represented six subgenera while four subgenera were identified in Sergentomyia. Different sand fly species identified in the present study consist of Phlebotomus (Larroussius) orientalis, P. (Anaphlebotomus) rodhaini, P. (Synphlebotomus) martini, P. (Phlebotomus) bergeroti, P. (P.) papatasi, P. (P.) duboscqi, P. (Paraphlebotomus) alexandri, P. (Parvidens) lesleyae, P. (Parv.) heischi, Sergentomyia (Parrotomyia) africana, S. (Sergentomyia) schwetzi, S. (S.) antennata, S. (S.) bedfordi group, S. (S.) dubia, S. (Sintonius) clydei, S. (Sin.) adleri, S. (Sin.) calcarata, S. (Sin.) subtilis, S. (Sin.) adami, S. (Sin.) satti, S. (Sin.) christophersi, S. (Sin.) capensis, S. (Sin.) thomsoni, S. (Sin.) affinis, and S. (Grassomyia) squamipleuris.In total, 100,772 sand fly specimens, belonging to twenty-five species  followed by P. lesleyae (0.14%). While the other Phlebotomus species constituted only 0.31% of the entire sand fly collection. Among the genus Sergentomyia, S. africana (59.1%) was the most prevalent species followed by S. schwetzi (5%).The relative abundance of these species is indicated in Table P. orientalis collected from the three sampling villages  using CDC light traps and sticky traps during the entire collection period are illustrated in Table P. orientalis captured per CDC trap/night in the three sampling villages . Likewise, the sampling villages significantly differed in their sand fly productivity on sticky trap collections . In both trapping methods, Ademeyti and Lemlem were the most productive sampling villages for P. orientalis population compared to Mentebteb  followed by peri-domestic (mean = 3.66 ± 1.48/m2 trap/night) and indoors (mean = 0.05 ± 0.02/trap/night) .Unlike light trap collections, habitat types had significant effects on the abundance of P. orientalis and other sand flies found resting at 6:00 to 8:00 hours in rooms. Of 3,054 indoor resting sand flies captured in the pyrethrum spray collections during 578 house visits, 48 (1.57%) P. orientalis were found resting inside houses. In particular, female populations of P. orientalis had low endophilic behavior, constituting only 0.95% of the entire indoor resting specimens collected. Among Sergentomyia spp. S. africana had a greater proportion (52.72%) followed by S. schwetzi . P. orientalis was abundant outdoors and had low abundance indoors with a ratio of 79:1 on m2 of sticky traps  from indoors and 15,901  from outdoors were captured on sticky traps. The result of the outdoor  ps Table .Table 6The annual mean maximum and minimum temperatures were 38.4°C in March and 15.8°C in January, while relative humidity was 63.5% in August and between 38-39% during the months of January to April 2012, respectively . P. orientalis showed distinct seasonality, with the greatest overall abundance between January and June, reaching its peak density (6.18 ± 2.43/trap-night) in March when the average temperature was also high (30.4°C). From July to December, including the rainy season (July-September) and shortly after that (October-December), there was a sharp decrease in abundance of P. orientalis  while the lowest was during August to October, which ranged from 0.07 to 0.27/m2 trap/night . In contrast, the mean monthly abundance of P. orientalis was correlated negatively with relative humidity  and rainfall (r = −0.467), respectively though the relationship with the later was not significantly different (P = 0.126). Mean monthly density of P. orientalis on sticky traps had a positive correlation with temperature , and was associated negatively with relative humidity  and rainfall .In light trap catches, monthly abundance of P. orientalis, the sex ratio in light traps was 2.1:1, which did not show any significant difference between sexes  as opposed to a very high ratio of male to female (12.7:1) on the sticky traps that was clearly significant (P = 0.000).Sex ratios  for different sand fly species demonstrated that males caught by all methods was higher than that of females , with an overall sex ratio of 1.3:1. For P. orientalis males with un-rotated or partially rotated genitalia during the study period . However, no freshly emerged P. orientalis males were recorded with other collection methods used.Sticky traps intercepted 655 od Table . Of thesP. orientalis captured between horizontally (HSTs) and vertically (VSTs) placed sticky traps . A relatively higher mean density of P. orientalis was found on horizontally placed sticky traps (mean = 60 ± 14.56/m2/night) than vertically deployed  and sixteen species of Sergentomyia (four subgenera). The sand fly fauna in the area is composed of the Afrotropical elements [In the remote rural villages of Tahtay Adiyabo district, entomological studies revealed the presence of twenty five species of phlebotomine sand flies, including nine species of elements ,16,17.Phlebotomus caught, P. orientalis was the most prevalent, accounting for more than 98%. This species is the proven vector of L. donovani, the causative parasite of VL, in Sudan, South Sudan, south western Ethiopia [et al., unpublished data). The preponderance of this species in sand fly catches was also noted in previous studies in north-west Ethiopia [P. martini and P. rodhaini were also recorded in the present study: the former being the major vector of VL in southern Ethiopia [of L. donovani between animal reservoir hosts in Sudan [P. papatasi, P. bergeroti and P. duboscqi) [L. major from blood samples was reported in this area [Sergentomyia spp., S. africana was found to be the predominant species (77.5%) followed by S. schwetzi (6.6%). In general, the species composition of phlebotomine sand flies encountered in the present study concord with previous reports in other parts of Ethiopia [Out of the nine species of Ethiopia ,18 and aEthiopia ; GebresiEthiopia ,20 as weEthiopia -23. P. mEthiopia  and the in Sudan . The thruboscqi)  were alshis area . Among tEthiopia ,23,25,26P. orientalis was collected in agricultural fields than other habitats. Agricultural fields are mostly vertisols, which are characterized by high contents of clay minerals that enhance swelling when hydrated and shrinkage upon desiccation, thereby, causing extensive cracking during the dry season [P. orientalis in cultivated fields than other habitats observed. Secondly, the presence of trees and scrub vegetations provide shade and source of sugar for adult populations [P. orientalis is mainly associated to forest area with large expanses of vertisols [Habitat preferences of most sand fly species is associated with biotopes, harboring high levels of stable mild temperatures and high humidity, and that contain decaying organic matter allowing better breeding sites and more suitable diurnal resting shelters ,28. In ty season , possiblulations . Earlierertisols ,30,31 anP. orientalis exhibited strong exophilic behavior expressed in outdoor/indoor index of 79:1 on m2 of sticky traps. Supportive of the sticky trap indoor collections, less proportion of P. orientalis females were found resting indoors in 578 house visits, indicating lower rates of endophily. Elnaiem et al. [P. orientalis in some villages of north-west Ethiopia also yielded less capture . As the weather, in Tahtay Adiyabo is typically warm from January through May, the people usually sleep outdoors in their compounds during these months. Farmers also usually keep domestic animals in the yard overnight often within a few meters of the sleeping area, a practice that contributes to an increased abundance of P. orientalis females. Therefore, these activities expose people to the bite of P. orientalis leading to an outdoor transmission of L. donovani.Exophilic and endophilic behaviors of sand flies are important from the control point of view and determining where transmission takes place. It is evident from the study that m et al.  reportedP. orientalis in Tahtay Adiyabo appear to increase in density during the hot-dry period of March to April. A similar trend was also reported in Sudan, where the numbers of P. orientalis captured using sticky traps and CDC light traps remained low in the early dry season (January and February) and increased highly between March and May, after the hot, dry weather has begun [P. orientalis appeared to reach its peak during the driest months of March and April in Kafta-Humera, north-west Ethiopia [L. donovani in the area . The current study also demonstrated that the adult populations of P. orientalis diminish and disappear as the rain commences. This population disappearance was also observed in preceding studies in southern Sudan [Our data demonstrated that as begun ,20,32,33Ethiopia . Importarn Sudan . Howeverrn Sudan  and in ern Sudan .P. orientalis abundance. Sharp increases in population density of P. orientalis from March to April coincide with an increase in temperature and a reduction in relative humidity in the area. Presumably, the rapid decrease in abundance of P. orientalis through July to October largely attributed to an increased amount of rainfall that completely floods the surface and seals the deep cracks of vertisols, leading to a micro-climate change in the breeding/resting sites of the fly population. Moreover, changes in the monthly temperature and relative humidity during those periods might have resulted in a decrease in population abundance. During the rainy season, P. orientalis seems to undergo diapauses as larvae, which were observed in the fourth larval stage of laboratory colony [Sand fly population dynamics is largely regulated by a complex interplay between the biotic potential of the vector species and meteorological-environmental conditions -37. In ty colony . The diaP. orientalis had higher proportion of male to female both in CDC light traps and sticky traps, though the mean number ratio of male to female for the former was not significant. This male biased sex ratio was also observed for other sand fly species [et al. [As for the sex ratio of sand flies throughout the collections,  species ,39,40. FP. orientalis. This habitat constituted more than 70% of P. orientalis immature males caught with either un-rotated or partially rotated genitalia. More accurate recording of the condition of males with un-rotated external genitalia can indicate the proximity of immature sites [P. orientalis in our study area [The natural breeding habitats of sand flies could be determined from sticky trap catches as these traps usually capture sand flies by passive interception rather than attraction in their diurnal/breeding habitats . In the re sites . Moreovere sites ,44. Deepudy area . AccordiP. orientalis was recorded on horizontally placed (HSTs) STs than vertically (VSTs) deployed. Both sexes of P. orientalis were attracted to HSTs, albeit twenty two times as many males as females were trapped. Interestingly, deploying STs horizontally can be a practical solution for monitoring sand fly populations, particularly in flat plains with crevice although it remains to be determined in other sand fly habitats. The higher proportion of male P. orientalis on HSTs in the present study could be due to the attraction behavior of this vector species to the horizontal surface, establishing mating swarms [Significantly, higher mean density of g swarms ,45. AlteP. orientalis was found to be the most abundant Phlebotomus species, showing distinct seasonality that mainly peaks during the dry season (March to April) within the entire study area. Furthermore, this study depicts the exophilic behavior of P. orientalis. This behavior is of practical importance because it apparently makes the species less vulnerable to insecticide residual spraying (IRS) or long lasting insecticidal nets in indoor conditions. However, the small number of P. orientalis collected in sticky traps and pyrethrum spray catch indoors may still be of epidemiological significance. Therefore, control programs designed to contain VL transmission in different villages of Tahtay Adiyabo should focus mainly on targeting P. orientalis in outdoors without ignoring its minor endophilic behavior.In conclusion, our study demonstrated that


Candida glabrata is a major opportunistic human fungal pathogen causing superficial as well as systemic infections in immunocompromised individuals and several other patient cohorts. C. glabrata represents the second most prevalent cause of candidemia and a better understanding of its virulence and drug resistance mechanisms is thus of high medical relevance. In contrast to the diploid dimorphic pathogen C. albicans, whose ability to undergo filamentation is considered a major virulence trait, C. glabrata has a haploid genome and lacks the ability to switch to filamentous growth. A major impediment for the clinical therapy of C. glabrata infections is its high intrinsic resistance to several antifungal drugs, especially azoles. Further, the development of antifungal resistance, particularly during prolonged and prophylactic therapies is diminishing efficacies of therapeutic interventions. In addition, C. glabrata harbors a large repertoire of adhesins involved in the adherence to host epithelia. Interestingly, genome plasticity, phenotypic switching or the remarkable ability to persist and survive inside host immune cells further contribute to the pathogenicity of C. glabrata. In this comprehensive review, we want to emphasize and discuss the mechanisms underlying virulence and drug resistance of C. glabrata, and discuss its ability to escape from the host immune surveillance or persist inside host cells. T. TC. albC. glabrata genomes [C. glabrata, although genes carrying minisatellites appear conserved. Remarkably, C. glabrata also harbors unusual types of minisatellites, so-called compound minisatellites with intermingled repeats and megasatellites containing long repeated motifs [EPA adhesion genes carry such repeats. A plausible hypothesis is that unusual minisatellites relate to high-frequency chromosomal rearrangements. This would further diversify expression as well as function of adhesion genes, which are considered important pathogenicity genes.These genome dynamics is also reflected in the high number of minisatellite sequences found in  genomes . Tandem  genomes ,64,65. Nd motifs . The evo7.C. glabrata. The core system is composed of four phenotypes identified on the basis of their colony color on plates containing CuSO4. They are called white (Wh), light brown (LB), dark brown (DB) and very dark brown (vDB) [C. glabrata are enigmatic, switching might be important in C. glabrata infections and host colonization.Two distinct morphologies, core and irregular wrinkled, which result from high-frequency phenotypic switching mechanisms, were recently discovered in wn (vDB) ,67. In awn (vDB) . These rwn (vDB) . The outwn (vDB) . Differe8.C. glabrata with the host innate immune cells include phagocytic cells [C. albicans destroys macrophages by switching to the hyphal growth while C. neoformans can lyse macrophages or escape via phagosomal extrusion [C. glabrata responds to host cell phagocytosis and how it can survive and persist inside the phagolysosome. The lack of morphogenesis does not allow physical killing of host cells by C. glabrata. Moreover, phagolysosome maturation brings a hostile environment for pathogens, including hydrolytic enzymes as well as a lower pH due to acidification [A. fumigatus lacking catalases normally degrading H2O2, shows attenuated virulence in a rat model of invasive aspergillosis [C. glabrata, a lack of CTA1, the gene encoding the only catalase, results in hypersensitivity to H2O2. Interestingly, C. glabrata strains show higher peroxide resistance than S. cerevisiae or C. albicans, suggesting a high intrinsic resistance to oxidative stress [C. glabrata in the phagolysosome, perhaps via autophagy, to help C. glabrata surviving in the nutrient-limited environment. Deletion of ATG11 or ATG17 results in defects in the reduction of peroxisomes and reduced survival upon phagocytosis. Thus, in addition to surviving ROS attacks, recycling of potential internal nutrients or the use of host-derived nutrients may be beneficial for the persistence and survival of C. glabrata in the host after phagocytosis.The first encounters of ic cells . Remarkaxtrusion ,72. To dfication ,74. In afication . Therefofication -77. For gillosis . For C. e stress . Indeed,e stress . Notably9.C. glabrata displays inherently high resistance to several antifungal drugs, limiting the efficacy of some antifungal drugs used in clinical therapy [For space constraints, we shall limit the discussion on antifungal drug resistance, but would like to refer to numerous excellent recent reviews on the use of antifungal drugs and the mechanisms of antifungal resistance in fungal pathogens ,79-81. IAzoles – These compounds represent most widely used class of antifungal drugs and they have been used to treat fungal infections for several decades. The cellular target of the azoles is the lanosterol 14-α-demethylase, encoded by the ERG11 gene [Candida spp, C. glabrata shows an inherently reduced azole susceptibility. In addition, prolonged and prophylactic treatment with azoles often results in the emergence of clinically resistant C. glabrata strains. For C. albicans, one azole resistance mechanism is the overexpression or mutation of the azole target Erg11 [C. glabrata azole-resistant clinical isolates, neither overexpression nor ERG11 mutations seem to mediate resistance [C. glabrata azole resistance: Cdr1, Cdr2 (Pdh1) and Snq2 [G11 gene . Inhibitet Erg11 ,84. Howesistance -87. Howesistance -91. Threand Snq2 ,92,93.C. glabrata to azoles but the mechanisms behind remain unclear. The growth-inhibition by fluconazole is suppressed by the addition of serum to the medium, perhaps because C. glabrata can take up cholesterol from the serum under these conditions [C. glabrata [C. glabrata to azoles.Aus1, another fungal ABC transporter implicated in sterol uptake in yeast , may be nditions . In addiglabrata . The autPolyenes - Polyenes are fungicidal antifungals and have been used for more than 50 years. These substances intercalate into ergosterol-containing membranes (i.e. mostly plasma membrane), thereby forming pores which result in leakage of cellular components, collapse of ion and electrical gradients and ultimately lead to cell death -Cys(6) transcription factor Pdr1 controls the expression of at least three ABC transporters involved in azole resistance in C. glabrata [PDR1 result in a constitutively active transcription factor, and these are prevalent in the majority of azole-resistant clinical isolates. In addition, strains harboring constitutively active PDR1 alleles also show increased virulence in mice, even in the absence of azole treatment [C. glabrata. Notably, similar to yeast, Pdr1 may also mediate azole resistance in response to mitochondrial dysfunction in C. glabrata [Finally, several mechanisms are operating in glabrata ,86. Pdr1glabrata . As in treatment . These rglabrata -110.S. cerevisiae, deletion of RPD3 encoding a histone deacetylase also results in enhanced susceptibility to azoles. Furthermore, Rpd3 is required for up-regulation of an ABC transporter in response to cycloheximide [C. albicans with histone deacetylase inhibitors also strongly increases the sensitivity of fungal pathogens to different classes of antifungal agents [CDR1 and CDR2 upon fluconazole treatment is significantly reduced in the presence of the histone deacetylase inhibitor trichostatin A [C. glabrata and should be considered as potential future drug targets.Interestingly, chromatin modification may account for novel mechanisms mediating drug resistance in certain fungi. For example, in heximide . In addil agents ,113. Notstatin A . Hence, 10.C. glabrata is a very successful human pathogen, accounting for up to 25% of all clinical Candida infections. Although new insights concerning the molecular mechanisms mediating virulence are surfacing, many open questions concerning host adaptability and pathogenicity remain. Hence, the field needs as much as possible genome-wide and global approaches, whereby researchers can make use of tools such as a deletion collection, an overexpression collection or an epitope-tagged ORFome just to name a few tools that have been revolutionizing research in the non-pathogenic baker's yeast [C. albicans or Cryptococcus neoformans [S. cerevisiae, hoping for new insights. The genome organization show rather limited differences, some of which may suffice to explain the striking differences in virulence. However, the remarkable and nearly endless combinatorial complexity of genetic interactions [S. cerevisiae and C. glabrata.'s yeast -116. Impoformans ,118. Howoformans ,120 as wractions  will makHence, the future calls for integration of different disciplines, including mathematics and molecular approaches delivering quantitative data. The use of systems biology approaches such as predictive mathematical modeling of quantitative biological data will facilitate a better understanding of the complexity underlying fungal pathogenicity. Furthermore, diagnostic tools exploiting molecular methods will have to improve concerning both speed and reliability, since this will facilitate clinical therapy. Of course, the clinical day-to-day reality is always in need for new and more efficacious antifungal drugs once the persistent difficulties concerning the rapid and accurate diagnosis of fungal species causing diseases have been overcome. In addition to classical small molecule drugs, novel antibody-based approaches may aid both diagnostic and therapeutic approaches, and even vaccines are now entering stage as feasible approaches to cure or combat systemic fungal disease.


Arabidopsis thaliana symbionts  using a systems biology approach. We evaluated multiple potential factors of microbial community control: we sampled various wild A. thaliana populations at different times, performed field plantings with different host genotypes, and implemented successive host colonization experiments under lab conditions where abiotic factors, host genotype, and pathogen colonization was manipulated. Our results indicate that both abiotic factors and host genotype interact to affect plant colonization by all three groups of microbes. Considering microbe–microbe interactions, however, uncovered a network of interkingdom interactions with significant contributions to community structure. As in other scale-free networks, a small number of taxa, which we call microbial “hubs,” are strongly interconnected and have a severe effect on communities. By documenting these microbe–microbe interactions, we uncover an important mechanism explaining how abiotic factors and host genotypic signatures control microbial communities. In short, they act directly on “hub” microbes, which, via microbe–microbe interactions, transmit the effects to the microbial community. We analyzed two “hub” microbes (the obligate biotrophic oomycete pathogen Albugo and the basidiomycete yeast fungus Dioszegia) more closely. Albugo had strong effects on epiphytic and endophytic bacterial colonization. Specifically, alpha diversity decreased and beta diversity stabilized in the presence of Albugo infection, whereas they otherwise varied between plants. Dioszegia, on the other hand, provided evidence for direct hub interaction with phyllosphere bacteria. The identification of microbial “hubs” and their importance in phyllosphere microbiome structuring has crucial implications for plant–pathogen and microbe–microbe research and opens new entry points for ecosystem management and future targeted biocontrol. The revelation that effects can cascade through communities via “hub” microbes is important to understand community structure perturbations in parallel fields including human microbiomes and bioprocesses. In particular, parallels to human microbiome “keystone” pathogens and microbes open new avenues of interdisciplinary research that promise to better our understanding of functions of host-associated microbiomes.Plant-associated microorganisms have been shown to critically affect host physiology and performance, suggesting that evolution and ecology of plants and animals can only be understood in a holobiont (host and its associated organisms) context. Host-associated microbial community structures are affected by abiotic and host factors, and increased attention is given to the role of the microbiome in interactions such as pathogen inhibition. However, little is known about how these factors act on the microbial community, and especially what role microbe–microbe interaction dynamics play. We have begun to address this knowledge gap for phyllosphere microbiomes of plants by simultaneously studying three major groups of  Microbial interactions between kingdoms are responsible for significant microbiome variation on the surface of plants. Highly connected microbes are most important, amplifying abiotic and host factors to cause large perturbations in the structure of microbial communities. Under natural conditions, plant growth and behavior strongly depend on associated microbial communities called the microbiome. Much research has been performed to evaluate how the environment and plant genes help to determine the structure of the microbiome. Here, we show that interactions between microorganisms on plants can be responsible for large portions of observed microbial community structures on leaves. Importantly, particular microbes, termed “hub microbes” due to their central position in a microbial network, are disproportionally important in shaping microbial communities on plant hosts. We discovered fungal and oomycete hub microbes that act by suppressing the growth and diversity of other microbes—even across kingdoms—and several candidate bacterial hubs, which largely positively control the abundance of other bacteria. We also showed that factors impacting the microbial community—such as plant genotype—are strongest if they affect colonization of a hub microbe because the hub in turn affects colonization by many other microbes. Our results further suggest that hub microbes interact directly or via the microbial community. Hub microbes are thus promising targets for better understanding the effects of host genomic engineering and for future work in controlling disease-associated and beneficial host-associated microbial communities. Hosts and their associated microbial communities are increasingly seen as inseparable entities (metaorganisms) whose ecology and evolution are inseparably entwined ,2. For eGiven the broad range of microbes that colonize above-ground parts of plants such as bacteria, yeasts, filamentous fungi , and proGenerally, three mechanisms contribute to microbial community structures: random colonization; species sorting by local factors ; and isolating factors such as dispersion and distance ,18. PrevPhyllosphere colonization proceeds via mechanisms that fundamentally alter the host, since some microbes participate in what can be described as niche construction. For example, many symbionts (including pathogens) deliver effector proteins to suppress, activate, or alter host defense ,28, and A. thaliana phyllosphere microbiomes. A systems biology approach documented highly interactive “hub” microbes, and in controlled laboratory experiments we confirmed that one, Albugo laibachii, strongly affects phyllosphere communities and found evidence for direct interactions by a second, Dioszegia sp. The results demonstrate that hub microbes mediate between sorting factors and microbial colonization, effectively amplifying sorting effects in the phyllosphere and stabilizing populations of specific microbes on individual plants. Our findings provide insights into the complexity of multikingdom interactions in the phyllosphere and improve the understanding of the dynamics of plant microbiome colonization.Most studies implicitly assume that abiotic and host factors differentiate microbial communities because of variable microbial adaptations. Research in the animal field has shown that for example, variation in the Major Histocompatibility class II (MHC) genotypes contributes to microbial variation among hosts . In humaA. thaliana populations that have been studied for several years Albugo, wd genera ,49.Albugo was reduced (in the CG experiment) or eliminated (in lab experiments) due to  resistance in the accession A. thaliana Ksk-1. We also observed much lower endophytic bacterial and fungal diversity than epiphytic caused by the “gateway” between the leaf surface (epiphytes) and interior (endophytes). This level of sorting occurs since endophytes and pathogens need to specialize and coevolve with hosts .Raw sequence data is publicly available online through MG-RAST project number 13322 , and the Tübingen wild sites: ERG, EY, JUG, PFN, WH .(TIF)Click here for additional data file.S5 FigLegends are common for the barcharts in the figure. Data is based on relative abundance calculated from data that was not first subsampled. Key: CG: Cologne garden experiment , and the Tübingen wild sites: ERG, EY, JUG, PFN, WH .(TIF)Click here for additional data file.S6 FigLegends are common for the barcharts in the figure. Data is based on relative abundance calculated from data that was not first subsampled. Key: CG: Cologne garden experiment , and the Tübingen wild sites: ERG, EY, JUG, PFN, WH .(TIF)Click here for additional data file.S7 FigAlbugo sp. was observed on the sample.Plotted points are scaled to measured endophytic Hpa . The color of the plotted points corresponds to whether or not “white rust” caused by (TIF)Click here for additional data file.S8 FigA. thaliana Ws-0, C: A. thaliana Col-0, K: A. thaliana Ksk-1. Yellow: Albugo sp. spores physically removed from inoculum by filtering, Green: Susceptible plants inoculated with Albugo sp. and associated microbes, Red: Resistant plants inoculated with Albugo sp. and associated microbes.W: (TIF)Click here for additional data file.S9 FigA. thaliana accession Ksk-1 carries an allele for resistance to Albugo sp., which lent partial resistance to the wild pathogen strains. Letters indicate significance at p < 0.1 (Tukey’s HSD).The (TIF)Click here for additional data file.S10 FigA. The correlation network, which is identical to that presented in (TIF)Click here for additional data file.S11 Fig2 > 3 is a cutoff we designed that accounts for distribution of the microbes and the strength of the correlation  for three measures of node (taxa) centrality, where yellow highlights taxa that are significantly more central in the network using a specific cutoff. ratio of abundance sum to maximum abundance (STM) x R Methods  for deta(TIF)Click here for additional data file.S12 FigA. thaliana. The first control were Albugo-free containing microorganisms that were associated with each Albugo strain but no Albugo spores. This control represents an abiotic factor, such as a distribution limitation, that in nature would limit the growth of Albugo. We tested both filter removal of Albugo spores Click here for additional data file.S13 Figt test, p < 0.05). Key: Ws-0: A. thaliana Ws-0, Col-0: A. thaliana Col-0, Ksk-1: A. thaliana Ksk-1. Green: Susceptible hosts, Red: Resistant hosts, Yellow: Filter removal of Albugo on all hosts.Results demonstrate that Albugo-infected plants had significantly lower bacterial diversity than controls (lines indicate (TIF)Click here for additional data file.S14 FigAlbugo sp. was confirmed regardless of the mechanism of removal of Albugo (see Albugo spores by filtering). Solid black lines are placed to facilitate comparison of control samples to Albugo-infected samples. W: A. thaliana Ws-0, C: A. thaliana Col-0, K: A. thaliana Ksk-1. Green: Susceptible hosts, Red: Resistant hosts, Yellow: Chemical inhibition of Albugo on all hosts by metalaxyl and benalaxyl.A strong suppressive effect on bacterial alpha diversity by (TIF)Click here for additional data file.S15 FigAlbugo-infected plants were more similar between experimental replicates than controls . Key: Ws-0: A. thaliana Ws-0, Col-0: A. thaliana Col-0, Ksk-1: A. thaliana Ksk-1. Green: Susceptible hosts, Red: Resistant hosts, Yellow: Filter removal of Albugo on all hosts. (S1_Data.xlsx)Results demonstrate that the final microbial communities recovered from (TIF)Click here for additional data file.S16 FigAlbugo-infected A. thaliana was more similar to other infected plant communities than to uninfected controls . For simplicity, not every possible comparison is shown. Key: Ws-0: A. thaliana Ws-0, Col-0: A. thaliana Col-0, Ksk-1: A. thaliana Ksk-1. Green: Susceptible hosts, Red: Resistant hosts, Yellow: Filter removal of Albugo on all hosts.Results demonstrate that bacterial communities at the order level from (TIF)Click here for additional data file.S17 FigA. thaliana leaves infected with Alb. candida Nc2 . Controls with inhibited spores used the oomycete-specific inhibitory compounds metalaxyl and benalaxyl.Results demonstrate high counts of bacteria on (TIF)Click here for additional data file.S18 Figt test with p < 0.05.For this analysis, bacterial reads identified as cyanobacteria at only the phylum level (with no more specific taxonomic assignment) have been removed. Indicated significant differences are based on a (TIF)Click here for additional data file.S19 Figt test, p < 0.05) on plants infected with Alb. candida compared to both controls. B. Genera without statistically significant  enrichment on two host accessions infected with Alb. laibachii. Some abundant taxa were not enriched due to Alb. laibachii infection, while others were only enriched on Alb. thaliana Ws-0 (the accession on which stock infections were kept) or were similar abundance in all treatments.A. Genera with significant increase or decrease ((TIF)Click here for additional data file.S20 FigDioszegia growth with bacteria (letters indicate significant differences at Tukey’s HSD p < 0.05). Dioszegia growth was significantly negatively affected by Rhodococcus sp. and Agromyces sp. compared to growth alone. B. Bacterial growth alone or with Dioszegia . The growth of Rhodococcus sp. was slightly increased in the presence of Dioszegia compared to growth alone. The strongest effect was on Caulobacter sp. where strong growth was completely inhibited by Dioszegia sp. Details about bacterial isolates are provided in Rhodococcus sp. interactions were not observed in the network analysis, but this was a lab isolate and the genus was very low abundance in the field.A. (TIF)Click here for additional data file.S21 FigAlbugo sp., W: A. thaliana Ws-0, C: A. thaliana Col-0, K: A. thaliana Ksk-1, Green: Susceptible host, Red: Resistant host, Yellow: filter removal of Albugo sp. on all hosts.Legends are common for the barcharts in the figure. Data is based on relative abundance calculated from data that was not first subsampled. Key: Spores + microbes: inoculation of Albugo sp. and associated microorganisms, Microbes only: inoculation of associated microorganisms after filter removal of (TIF)Click here for additional data file.S22 FigAlbugo sp. and Dioszegia sp. reinforces their putative role based on paired microbe correlations in limiting abundance of many epiphytic bacterial genera.Interkingdom correlations reveal several genera correlated significantly to fungal and bacterial epiphyte and endophyte diversity. Strong negative correlations of (TIF)Click here for additional data file.S23 FigAlbugo sp., W: A. thaliana Ws-0, C: A. thaliana Col-0, K: A. thaliana Ksk-1, Green: Susceptible host, Red: Resistant host, Yellow: filter removal of Albugo sp. on all hosts.Legends are common for the barcharts in the figure. Data is based on relative abundance calculated from data that was not first subsampled. Key: Spores + microbes: inoculation of Albugo sp. and associated microorganisms, Microbes only: inoculation of associated microorganisms after filter removal of (TIF)Click here for additional data file.S24 FigThis effect was especially apparent in the V5/V6/V7 region where sampling was deepest. Data is based on observed number of bacterial genera and each line represents one sample.(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Tablep < 0.01).Enrichment at a location is based a significantly higher relative abundance there compared to any other location (Tukey’s HSD (DOCX)Click here for additional data file.S4 Tablep < 0.01).Enrichment in fall or spring is based on a significantly higher relative abundance in that season compared to the other season (Tukey’s HSD (DOCX)Click here for additional data file.S5 Tablep < 0.05).Enrichment on an accession is based a significantly higher relative abundance on that accession compared to any other accession (Tukey’s HSD (DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Picris subsect. Hieracioides with major focus on the widely distributed P. hieracioides and its closely related congeners, P. hispidissima, P. japonica, P. olympica, and P. nuristanica. Accessions from 140 sample sites of the investigated Picris taxa were analyzed on the infra- and the inter-specific level using nuclear (ITS1-5.8S-ITS2 region) and chloroplast (rpl32-trnL(UAG) region) DNA sequences. Genetic patterns of P. hieracioides, P. hispidissima, and P. olympica were shown to be incongruent and, in several cases, both plastid and nuclear alleles transcended borders of the taxa and genetic lineages. The widespread P. hieracioides was genetically highly variable and non-monophyletic across both markers, with allele groups having particular geographic distributions. Generally, all gene trees and networks displayed only a limited and statistically rather unsupported resolution among ingroup taxa causing their phylogenetic relationships to remain rather unresolved. More light on these intricate evolutionary relationships was cast by the Bayesian coalescent-based analysis, although some relationships were still left unresolved. A combination of suite of phylogenetic analyses revealed the ingroup taxa to represent a complex of genetically closely related and morphologically similar entities that have undergone a highly dynamic and recent evolution. This has been especially affected by the extensive and recurrent gene flow among and within the studied taxa and/or by the maintenance of ancestral variation. Paucity of phylogenetically informative signal further hampers the reconstruction of relationships on the infra- as well as on the inter-specific level. In the present study, we have demonstrated that a combination of various phylogenetic analyses of datasets with extremely complex and incongruent phylogenetic signal may shed more light on the interrelationships and evolutionary history of analysed species groups.We investigated genetic variation and evolutionary history of closely related taxa of  Identifying patterns of genetic variation within and among closely allied congeners is crucial in understanding their evolutionary relationships and processes involved in their diversification. Exploration of genetic patterns across the entire geographic ranges of each of the constituent species not only enables clarification of modes of their diversification and historical changes in spatial distribution, but may also shed light on their potential reciprocal interactions (e.g. hybridisation and introgression).In many instances, however, multi-gene phylogenies of closely related and recently diversified taxa are incongruent, often with alleles transcending species or genetic groups, and finally cause the genetic patterns to exhibit a diffusive or mosaic-like structure Studies that have carefully and explicitly analyzed multiple reasons of genetic incongruence are not common Picris subsect. Hieracioides Vassiliev (Compositae) Picris subsect. Hieracioides comprises seven morphologically very similar taxa from Europe, Asia and Africa: P. abyssinica Sch. Bip., P. hieracioides L. including subspecies P. hieracioides subsp. umbellata (Schrank) Ces. and P. hieracioides L. subsp. hieracioides (referred as P. h. subsp. umbellata and P. h. subsp. hieracioides in the text), P. hispidissima (Bartl.) W. D. J. Koch, P. japonica Thunb., P. nuristanica Bornm., and P. olympica Boiss Picris subsect. Hieracioides is still limited. Only three taxa from the subsection Hieracioides, namely P. abyssinica, P. hieracioides and P. nuristanica, were marginally analysed in a study focused on the genus Leontodon. The two latter Picris species were shown to be closely related, while P. abyssinca was genetically much more distant and clustered together with other North African Picris taxa Hieracioides are reported to be perennials with homocarpic achenes  and two-hooked anchor hairs on the stem and peduncles P. hieracioides also to P. hispidissima, P. olympica and marginally also to P. japonica and P. nuristanica.Members of P. hieracioides, P. hispidissima, P. japonica, and P. nuristanica revealed that these taxa are very similar morphologically and karyologically as well as they are closely related genetically (AFLP), their evolutionary relationships and mutual interactions were left unresolved P. hispidissima and P. hieracioides and also found populations that represented genetic admixtures of different P. hieracioides subspecies and/or genetic groups, mostly accompanied by transitional morphology. In all cases, the observed intermediate genetic and/or morphological states were hypothetically attributed to hybridisation although this heterogeneity may be also a result of retention of ancestral variation. No studies collecting the relevant data on P. olympica have been done prior to the present study. Likewise, overall relationships within Picris subsect. Hieracioides have not been investigated yet. Thus the specific goals of the present study are:Although our previous investigations on P. hieracioides, P. hispidissima, P. japonica, P. olympica, and P. nuristanica.Clarifying the phylogenetic relationships among P. hieracioides in Europe and Asia Minor, the distribution of its infraspecific genetic groups, and comparing its genetic variation with that of P. hispidissima and P. olympica.Exploring the overall genetic pattern of Shed more light on potential interactions within the studied group and on the processes acting during evolutionary history.Picris hieracioides is a biennial to perennial, morphologically highly variable species that includes two infraspecific taxa, P. h. subsp. umbellata and P. h. subsp. hieracioides. The subspecies of P. hieracioides are morphologically distinguishable by a combination of several morphological traits on both vegetative and reproductive organs. This was definitely proved also by cultivation experiments under environmentally homogeneous conditions Picris hispidissima is a biennial species  that differs morphologically from P. hieracioides in having a pectinate-ciliate indumentum of involucral bracts and inflation of the peduncle P. olympica, is a perennial characterized by a low caespitose growth with quite short ascending scapose-like stems bearing only a few capitula. It is considered to be morphologically closely related to P. hieracioidesP. hieracioides being the most widespread (distributed across the major part of Europe and Asia Minor) and having wide ecological amplitude P. h. subs. hieracioides is mainly biennial, growing on open, dry, xerothermous biotopes with no bedrock preference and thriving in lowlands or lower mountains throughout all of Europe. In contrast, P. h. subsp. umbellata is a short-lived perennial herb inhabiting humid tall herb communities from montane forests to sub-alpine communities across European calcareous mountain ranges. Both P. hispidissima and P. olympica have restricted distributions. The former occurs in dry, calcareous rocky slopes and crevices in the Adriatic coastal mountains from Croatia to Montenegro, while the latter is restricted to a few high mountain ranges in western Anatolia where it inhabits open, craggy grasslands with alkaline bedrocks at alpine levels P. hieracioides, have spread from their natural biotopes into man-made habitats and migrated along anthropogenic corridors  and P. nuristanica (1 population). We did not include samples of P. abyssinica since it was proved that it is genetically highly divergent from P. hieracioides and P. nuristanica but is more closely related to other African taxa rpl32-trnL(UAG) regions  Eig., P. capuligera (Durieu) Walp., P. pauciflora Willd., P. rhagodioloides (L.) Desf., P. scaberrima Ten., P. sinuata (Lam.) Lack, and P. strigosa M. Bieb.) and two members of the closely related genus Helminthotheca (H. aculeata (Vahl) Lack and H. echioides (L.) Holub). Voucher specimens were deposited in the herbarium of the Institute of Botany at the Slovak Academy of Science (SAV).Selection of appropriate outgroup taxa was based on previously published studies rpl32-trnL(UAG)trnV(UAC)-ndhC, ndhF-rpl32 and trnH(GUG)-psbA) We utilised sequences of the nuclear ribosomal internal transcribed spacer (ITS1-5.8S-ITS2 region) and the non-coding cpDNA intergenic spacer rpl32-trnL(UAG) spacer (referred as cpDNA henceforth in the text) were amplified using the standard PCR reaction, employing the following universal primer pairs: P1A, P4 and internal ones, ITS2 and ITS3, if necessary trnL-retF and rpl32-retRTotal genomic DNA was extracted from silica gel-dried leaf tissue by the DNeasy plant mini kit , following the manufacturer's protocol. The ITS region and the plastid Pfu polymerase , 0.2 mM of each dNTP, 0.2 µM of each primer, 1 µL of DNA template, and reaction buffer containing 2 mM of MgSO4 . For ITS amplification, we used the following thermocycler program: preheating at 94°C for 3 min, then running it for 35 cycles at 94°C for 30 sec, then at 50°C for 30 sec, and 72°C for 60 sec, with the final extension at 72°C for 10 min, and cooling at 4°C. The cpDNA region was amplified using the “rpl16” program, as described by xl sequencer at BITCET Consortium, Comenius University in Bratislava. The sequencing of a few accessions repeatedly failed for the ITS region and these were therefore excluded from further analyses , using PCR reaction volumes of 25 µL consisting of 0.75 U of analyses .Sequences were edited and aligned manually using the BioEdit program version 7.0.4.1 P. hieracioides and also other taxa possessed homogenized ITS sequences. The geographic pattern of homogeneous and heterogeneous ITS sequences was obviously not random, indicating a genuine variation rather than erroneous signals caused by inaccurate amplification. Although the intra-individual ITS variation might be revealed by cloning procedure, this approach, on the other hand, may result in amplification of numerous ITS sequence types which do not represent relevant variability We believe that the observed intra-individual variation did not arise from PCR errors, because numerous accessions of Altogether we generated three alignments: the first one was designated as ITS_1 and involved 117 sequences without APS . ITS_1. Finally, the third cpDNA alignment comprised all plastid sequences, in particular 219 accessions . cpDNA. Indels were not coded separately but treated as missing data. The aligned datasets were analysed independently using the following phylogenetic approaches:Maximum parsimony (MP) phylogenetic analysis was performed with the heuristic search option in PAUP* version 4.0b10 Bayesian inference (BI) was run in MrBayes version 3.1.2, using the Markov Chain Monte Carlo algorithm (MCMC) th generation. The first 25% of sampled trees were regarded as ‘burn-in’ trees and were discarded prior to reconstruction of a 50% majority-rule consensus tree. Stationarity was confirmed by checking convergence diagnostic parameters. Specifically, the average standard deviation of split frequencies was lower than 0.01 in all cases; the plots of generations versus log probability of the observed data showed no obvious trends; and the Potential Scale Reduction Factor (PSRF) approached 1. Finally, topologies and node posterior probability values were compared among the runs. The topologies were stabilized among all datasets with only minute differences in branching pattern of terminal clades. Nodes with posterior probability (PP) values of 0.90 and above were regarded as significant and those with PP values below 0.90 regarded as non-significant.All BI analyses were run with four independent Metropolis-coupled MCMC chains (three heated and one cold chain) for ten to twenty five million generations and sampled every 10003. Net-like approaches were used to identify and display potential contradictory signals in the datasets. All alignments including ITS_2 containing intra-individual polymorphic sites were analysed using the neighbour-net analysis of 4. In order to precisely specify the amount of phylogenetically informative signal in the datasets, we analysed spectrum of supporting nucleotide positions. There are three groups of supporting positions recognized by Wägelle and Rödding th generation. Another MCMC analysis was run with settings suitable for a subsequent JML analysis (see below), i.e. with piecewise constant population size model and 40 million generations sampling every 1000th generation. The computer program Tracer version 1.5 5. We also apply a Bayesian coalescent-based approach to estimate a species tree employing *BEAST as implemented in the program BEAST version 1.7.4 P. olympica within the subsection Hieracioides; sister relationship of P. nuristanica and P. japonica; and monophyly of P. hispidissima and P. hieracioides , appeared to be a part of ingroup. In contrast, all accessions a priori considered as outgroup taxa were shown to be part of real outgroup in the plastid region analysis (see below). Thus, sequences of 11 accessions (7 species) are considered outgroup for the ITS region while 14 accessions (9 species) for the cpDNA spacer. Information on the DNA datasets and details on maximum parsimony analyses are summarized in A total of 202 sequences of the ITS region and 205 of the cpDNA spacer of taxa a priori considered as ingroup were obtained. Results of phylogenetic analyses, however, revealed that beside of the 202 ingroup ITS sequences, another three accessions that were a priori considered as outgroup taxa  did not show the analysed ic group  and 2A. oup taxa  and 2A.hieracioides species, P. hispidissima, P. japonica, P. olympica, P. nuristanica, P. scaberrima, and P. strigosa, formed their own strongly supported sub-clades, with P. olympica and P. scaberrima being the most divergent .The Bayesian 50% majority-rule consensus tree and the strict consensus MP tree based on the ITS_1 dataset (117 sequences without APS) displayed essentially identical topologies with only small differences in terminal positions and/or in clade supports . All ingivergent . Picris ibutions  and 3: I blue in  and 3; Is red in  and 3; Ibrown in  and 3. WP. olympica separated from the rest by a set of long parallel and strongly supported edges; (2) lowland populations of P. hieracioides, morphologically corresponding to P. h. subsp. hieracioides, are clearly separated from all ingroup and outgroup taxa (ITS 1A and 1B). The exception represented few populations of the P. h. subsp. hieracioides morphotype that clustered together with populations attributable to the P. h. subsp. umbellata morphotype in an unresolved ITS4 group; (3) a large heterogeneous group associating several smaller well supported splits corresponding to P. hispidissima, P. japonica, P. nuristanica, and two lineages composed of mountainous populations of the P. h. subsp. umbellata morphotype (ITS_2 and ITS_3); and (4) a group of both morphotypes of P. hieracioides (ITS_4) and P. strigosa, a presumably outgroup species. Picris scaberrima formed a long edge linked with outgroup taxa by short parallel splits. Importantly, the second neighbour-net analysis based on the ITS_2 dataset (206 ITS sequences) revealed essentially the same pattern as the previous one (figure not shown). This apparently indicates that heterogeneous sequences did not significantly affect the overall split pattern. Individuals with un-homogenized ITS sequences and numerous APS appeared mostly in the central position of the network, while those possessing more homogenized sequences, with lower number of APS, were preferentially incorporated in terminal positions (figure not shown).The split decomposition diagram based on the ITS_1 dataset (117 ITS sequences) showed four main splits having various levels of bootstrap support : (1) P. Hieraciodes plus P. strigosa and P. scaberrima, are supported only by 7 nucleotide positions. Within the functional ingroup, the highest support obtained P. olympica (9 positions), P. japonica (4 positions), the P. hieracioides clade with lowland populations corresponding to the P. h. hieracioides morphotype (3 positions) and P. nuristanica (2 positions). All other above mentioned splits/subclades were supported at maximum by a single position.Analyses of spectrum of supporting positions revealed that majority of phylogenetically informative positions are shared by outgroup taxa . The entMP and BI analyses yielded trees with different topologies to a certain extent. However, there was no strongly supported incongruence between them. In order to maintain consistency in the presentation of our results we discuss only the topology based on the BI phylogenetic tree.P. scaberrima and P. strigosa appeared clustered together with other outgroup taxa. All accessions of the Hieracioides group appeared in a largely unresolved clade with five essentially supported cpDNA subclades and two unsupported groups (cpDNA_A–G) placed in a basal polytomy .The parsimony-based haplotype network revealed 34 very closely related ingroup haplotypes, mostly connected by one or two steps  and the p<0.001) incongruence between both gene trees, we performed concatenated analyses because our split spectrum analyses show that the observed discordances are not strongly supported. Thus, incongruence between both genetic regions could rather reflect paucity of phylogenetically informative signal . Non-hieracioides ingroup species, namely P. japonica, P. olympica and P. nuristanica, each clustered in strongly supported sub-clades, repeatedly with P. olympica being the most divergent . Importantly, although weakly supported, the majority of accessions assigned to morphotypes of both subspecies of P. hieracioides as well as to P. hispidissima tended to form taxon specific groupings . By contrast, 31 cases with p<0.05 were found in the cpDNA dataset involving accessions of the corresponding pairs of the following taxa: P. olympica vs. P. hieracioides, P. olympica vs. P. rhagodioloides; P. sinuata vs. P.capuligera; and H. aculeata vs. H. echioides . Thus, in spite of the complex patterns, we might assume the following: (1) the analysed members of the subsection Hieracioides have a monophyletic origin, with P. scaberrima and P. strigosa being their sister taxa; (2) P. olympica is basal within the subsection Hieracioides, i.e. it is sister to all other members of this subsection; (3) all Asian species are sister to the European ones; and (4) P. japonica + P. nuristanica are sister to P. hieracioides + P. hispidissima. Interrelationships among populations of P. hieracioides and P. hispidissima are left unresolved, however.Tree-building and network analyses of both the nuclear and plastid markers of the studied cies see , althougWhich factor(s) might be responsible for low resolution, varying supports and discordances between gene tree topologies? In the present study, we have shown by the split spectrum analysis that one of the primary and crucial factors responsible for this pattern is a paucity of parsimony informative and node supporting nucleotide positions. Deficiency of phylogenetically informative signal in the datasets analysed was unambiguously evidenced by the low number of primary nucleotide homologies  and 7B. Picris taxa P. hieracioides and P. hispidissima as well as the low nodal support could signalise either influence of this phenomenon or violation of prior assumption by hybridisation or other processes. Likewise, the disjunctive occurrence of the ancestral plastid haplotypes in populations of P. olympica and the spatially distant P. hieracioides might be the consequence of incomplete lineage sorting. By contrast, the mosaic pattern detected in the ITS dataset may repeatedly suggest influence of hybridization.Furthermore, incomplete lineage sorting represents another evolutionary force that might be responsible for the discordances and complex genetic patterns in the six Picris taxa might be indicated by the following facts. (1) Considerable intra-individual and intra-population ITS sequence heterogeneity and allele transcending clades and taxon borders in P. hieracioides, P. hispidissima, and P. olympica. On the other hand, maintenance of two or more ITS variants within a single genome might be attributed to other molecular-genetic processes P. hieracioides and P. hispidissima, which has been recently documented .Hybridisation might substantially influence phylogenetic reconstructions at even lower hierarchical levels. Decreased phylogenetic tree resolution, incongruent patterns in different gene trees, and occurrence of alleles transcending taxa or genetic groups are commonly observed in plant phylogenies in which extensive gene exchange between taxa has been documented Picris taxa are rather recent or have occurred later after speciation events. This hypothesis corresponds well mostly in the case of comparatively distantly related species, P. olympica with P. hieracioides, P. olympica with P. rhagodioloides, and P. capuligera with P. sinuata. Such hybridisation events might take part during the Pleistocene glacial/interglacial range shifts, especially, at sites where lowland taxa could have had recurrent contact with mountainous ones P. hieracioides as well as between P. hieracioides and P. hispidissima. One of the most plausible explanations might be that the hybridization events among those taxa happened just after their diversification. Especially in case of the two subspecies of P. hieracioides, the divergence of genetic lineages might be shallow causing hybridization to be undetectable Posterior predictive checking revealed hybridization/introgression at least in five pairs of the studied species. Four pairs were recorded exclusively by simulations on the cpDNA dataset and one pair was detected by simulations on both the plastid and the nuclear dataset. Since the method of posterior predictive checking is powerful unless the hybridization event has occurred very rapidly after the speciation event H. aculeata and H. echioides. The sample size might be the limitation factor in the posterior predictive checking approach. Since only a single individual per each aforementioned species (outgroup) was sampled, their population sizes cannot be properly estimated for species tree reconstruction. Therefore we assume this result might be a methodological artefact.The overall failure to detect hybridization events by simulations on the ITS dataset might be attributed to the presence of recombination and/or concerted evolution within the ITS locus. As stated in P. hispidissima, fit into the reticulate evolution scenario or, alternatively, its large genetic heterogeneity arose from genetic erosion. The latter hypothesis might be explained by recurrent introgression with adjacent populations of P. hieracioides after their diversification. We are more inclined to the latter scenario because all taxa are morphologically rather well delimited, inhabit ecologically specific biotopes, and trans-taxon alleles are concentrated predominantly in areas of sympatry or parapatry.Finally, it remains arguable whether some of the studied taxa, but especially Picris taxa from the subsection Heiracioides, it is highly tricky or even impossible to discern between these two processes. Reasons for this ambiguity might come from weak genetic divergence of analysed taxa, and importantly from presence of gene transfer via introgression which, moreover, most probably occurred repeatedly during their evolutionary history.Although methods to potentially distinguish between hybridisation and incomplete lineage sorting have been developed, there is still a high risk of confusing these two phenomena or they cannot be discerned in some cases at all P. hieracioides and P. hispidissima varied considerably across phylogenetic trees, with both concordant and discordant patterns being detected . Thus, the plastid pattern exhibited an apparently mosaic-like spatial structure and two or more species/genetic groups shared same plastid haplotypes. This was, especially, true for the sympatric/parapatric populations . ComplexPicris species was shaped by the interplay of several factors and evolutionary processes. Their diversification was most likely rapid, and may have occurred in an allopatric manner. Since all of these taxa, but especially, P. hispidissima and P. olympica, have unique habitat and ecology preferences , we presume that ecological factors also played an important role in their speciation.Our results suggested that evolution of the studied P. olympica and P. hispidissima as well as by the accumulation of rare haplotypes, lies in the southern European peninsulas and partly also the Alps and the Carpathians  P. h. subsp. hieracioides was located more southwards, especially, in lowland and hilly regions of the Apennine Peninsula and plausibly also in the Balkan Peninsula. Subsequently, they colonised other habitats of their present day occurrence in central and north-western Europe as well as in Asia Minor from these southern refugia.Furthermore, genetic lineages detected within Picris hieracioides has been here evidenced to be highly heterogeneous across both markers. Actually, both plastid and ITS nuclear alleles have transcended borders of both subspecies of P. hieracioides, as recently delimited in P. hieracioides displayed transitional morphological variation. In addition, from ecological point of view, P. hieracioides is characterised by an exceptionally large range of habitat preferences P. hieracioides are artificial biological units, representing rather ecotypes evolved recurrently in different evolutionary lineages under similar environmental influences than real taxa.An important taxonomic question is whether the analysed taxa should be recognised as distinct species/subspecies, or whether they rather represent informal phylogeographic lineages within a single heterogeneous taxon. The latter possibility has been favoured in case of several other complex plant species groups P. hieracioides, but also on cultivation experiments realised under environmentally homogeneous conditions P. hieracioides, originating from almost the entire studied area P. hispidissima retained its morphological distinctiveness under environmentally homogenous conditions . Thus, taxonomically important morphological features of these entities are not a consequence of environmental plasticity, but are genetically inherited instead. All three taxa are well defined morphologically, ecologically and partially also genetically (AFLP's) at least within ‘pure’ populations untouched by their recent mutual secondary contacts.Ecotype hypothesis, however, strongly contradicts results of our previous study focused not only on investigation of wild populations of P. hieracioides as well as P. hispidissima are recently diversified and thus genetically weakly separated taxa. Additionally, their genetic variation has been significantly affected by their reciprocal multiple secondary contacts, taking place after anthropogenically mediated range expansions. Due to the lack of habitat and breeding isolation barriers, hybridisation and introgression among these taxa occur frequently in contact zones. This process leads to the genetic and morphological homogenisation of affected populations, erasing their distinctness in contact zones. Similar cases of taxon fusion caused by hybridisation preceded by migration of populations enhanced by human activities have been reported for instance in members of the genus KnautiaCerasusP. hieracioides at currently accepted taxonomic ranks P. hispidissima should be included under highly variable P. hieracioides as separated subspecies: P. h. subsp. hispidissima (Bartl.) Slovák and Kučera, comb. nova, hoc loco .Both, subspecies of Although this study does not provide comprehensive and firm taxonomic conclusions, it represents an important background for future investigations on this intricate and dynamically evolving species complex. Last but not least, the present investigation documents rapid process of taxon erosion, happening due to the anthropogenic disturbance of natural biotopes.Table S1Locality details of studied taxa, number of individuals used for the particular molecular analyses, and GenBank accession numbers.(DOC)Click here for additional data file.Table S2Summary of nucleotide site variation for the nuclear ITS region in the studied taxa.(XLS)Click here for additional data file.Alignment S1ITS_1: comprised 117 ITS sequences without APS. The accession labels in alignments follow the population and taxon codes used in (TXT)Click here for additional data file.Alignment S2ITS_2: comprised all 206 ITS sequences. The accession labels in alignments follow the population and taxon codes used in (TXT)Click here for additional data file.Alignment S3cpDNA: comprised all 219 rpl32-trnUAG sequences.L The accession labels in alignments follow the population and taxon codes used in (TXT)Click here for additional data file.Alignment S4ITS_cpDNA: comprised 117 ITS sequences without APS and corresponding plastid ones. The accession labels in alignments follow the population and taxon codes used in (TXT)Click here for additional data file.


Trypanosoma cruzi, the etiological agent of Chagas disease. Infection is often subclinical, but severe disease has been described in both free-ranging and captive primates. Panstrongylus megistus, a major T. cruzi vector, was found infesting a small-primate unit at Brasília zoo (ZooB), Brazil. ZooB lies close to a gallery-forest patch where T. cruzi circulates naturally. Here, we combine parasitological and molecular methods to investigate a focus of T. cruzi infection involving triatomine bugs and Neotropical primates at a zoo located in the Brazilian Savannah.Neotropical primates are important sylvatic hosts of T. cruzi infection in vectors using optical microscopy (n = 34) and nested PCR (n = 50). We used quantitative PCR (qPCR) to examine blood samples from 26 primates and necropsy samples from two primates that died during the study. We determined parasite lineages in five vectors and two primates by comparing glucose-6-phosphate isomerase (G6pi) gene sequences.We assessed Trypanosoma cruzi was found in 44 vectors and 17 primates (six genera and eight species); one Mico chrysoleucus and one Saguinus niger had high parasitaemias. Trypanosoma cruzi DNA was detected in three primates born to qPCR-negative mothers at ZooB and in the two dead specimens. One Callithrix geoffroyi became qPCR-positive over a two-year follow-up. All G6pi sequences matched T. cruzi lineage TcI.T. cruzi transmission within a small-primate unit at ZooB – with vectors, and perhaps also parasites, presumably coming from nearby gallery forest. Periodic checks for vectors and parasites would help eliminate T. cruzi transmission foci in captive-animal facilities. This should be of special importance for captive-breeding programs involving endangered mammals, and would reduce the risk of accidental T. cruzi transmission to keepers and veterinarians.Our findings strongly suggest vector-borne The online version of this article (doi:10.1186/s13071-016-1334-7) contains supplementary material, which is available to authorized users. Trypanosoma cruzi, the etiological agent of Chagas disease, is among the most epidemiologically relevant ones [http://www.mammalparasites.org]. Chagas disease is endemic throughout Latin America, where about six million people are infected with T. cruzi . Lion tamarins (Leontopithecus spp.) can sustain long-lasting infections, often with high parasitaemias but with a relatively mild clinical picture, in the Brazilian Atlantic forest [Carlos Chagas was the first to describe experimental  from the Primatology Centre of Rio de Janeiro, Brazil, where transmission mediated by Panstrongylus megistus was suspected [T. cruzi and may develop severe Chagas disease [ America . Trypanorá state . Anti-T.uspected . Captive disease –22.T. cruzi is relevant in several respects. First, T. cruzi can kill valuable specimens including those belonging to endangered species; second, infection of laboratory primates can distort the results of animal-based research aimed at other ends; third, infected individuals in translocation-reintroduction programs can contribute to the spread of the parasite among free-ranging populations; finally, and importantly, infection can result in accidental transmission of the parasite to primate keepers, handlers, or veterinarians. Here, we combine parasitological and molecular methods to investigate a focus of T. cruzi infection involving triatomine bugs and Neotropical primates at a zoo located in the Brazilian Cerrado, where T. cruzi circulates extensively among wildlife and native vectors.Infection of captive primates with This study was approved by the institutional review board of the Institute of Biological Sciences, University of Brasília, Brazil (CEUA-UnB No. 155506/2013).T. cruzi cycles are common in the Cerrado [T. cruzi infection has been recorded in Didelphis albiventris [Brasília, Brazil’s capital city, lies within the Cerrado eco-region, a mosaic of savannahs, dry forests/shrubs, and gallery forests originally covering most of central Brazil. Enzootic  Cerrado . Brasíliiventris . The smaTrypanosoma cruzi infection was first investigated by optical microscopy (OM) in the bugs that arrived alive to the laboratory . We first amplified 188 bp from the T. cruzi nuclear repetitive satellite region with primers TCZ1 and TCZ2 [T. cruzi culture  was used as a positive control, and MilliQ water and DNA from lab-reared, uninfected triatomines as negative controls. PCR products were resolved in 1.3 % agarose gel, stained with ethidium bromide, and visualised using UV fluorescence.Triatomines collected in the ZooB small-primate unit were identified after Lent & Wygodzinsky . Trypanoand TCZ2 ; amplicoand TCZ2 ; see AddT. cruzi detection and quantification. Necropsy samples  from one Saguinus niger and one Callithrix penicillata that died during the course of the study were also investigated. DNA was extracted from blood samples using the WizardTM Genomic DNA Purification Kit (Promega), and from necropsy samples using the Mini Spin Plus Kit (Biopur). PCR reactions were first carried out with primers TCZ1 and TCZ2 as described above; the products of this PCR were diluted 1:60 in MilliQ water and 2 μL were used as template for real-time quantitative PCR (qPCR) with Power SYBR© Green chemistry (Applied Biosystems) and primers TCZ3 and TCZ4 [T. cruzi culture (108 parasites/mL) and serially diluted ten-fold to between 105 and 10−2 parasite. The standard curve relates qPCR threshold cycle values and known log-scale DNA concentrations [2 = 0.99.Twenty-six Neotropical primates were investigated, six of which were born at ZooB  gene was PCR-amplified as described in Brenière et al.   were found infected with T. cruzi  primates tested by qPCR  and one Saguinus niger   in the spleen of one C. penicillata and in the heart, spleen, and intestine of the highly parasitaemic S. niger mentioned above. Five out of six primates born at ZooB tested positive for T. cruzi DNA by qPCR, including three individuals born to qPCR-negative mothers .T. cruzi infection focus in triatomine bugs and captive Neotropical primates at Brasília zoo in central Brazil. Highly-sensitive molecular assays detected T. cruzi nuclear DNA in most of the vectors (88 %) and primates (65.4 %) we tested. Infection was identified in primates of six genera and eight species, including the endangered Leontopithecus chrysomelas, the vulnerable Saguinus niger, and species with unknown preservation status such as Mico chrysoleucus and Pithecia irrorata (see www.iucnredlist.org). Infection with T. cruzi is harmful to the primates and brings about a non-negligible risk of accidental transmission of the parasite to animal keepers, handlers, and veterinarians.We have presented a detailed description of a T. cruzi strain as P. megistus caught in their lodgings. This finding is strongly suggestive of within-cage, P. megistus-mediated parasite transmission. Although we did not test for anti-T. cruzi antibodies through serology, which might have revealed infection in qPCR-negative individuals [T. cruzi from triatomines either through direct contact of infected vector faeces with skin or mucosae or by the oral route when bugs carrying the parasite are eaten or contaminate foods or beverages [P. megistus nymphs we collected inside primate lodgings tested positive for T. cruzi  of endangered species such as the flagship lion tamarins –34. Prevparasite , 35, so ulations , 14; inf success . By showP. megistus colony within the small-primate unit at ZooB. Infected bugs were found inside primate wooden shelters resembling the hollow-tree vertebrate nests and refuges P. megistus occupies in the wild [P. megistus is primarily associated with the humid Brazilian Atlantic forest [T. cruzi (TcI lineage) has been shown to circulate [Panstrongylus megistus, one of the most important vectors of human Chagas disease, can also colonise in and around human dwellings [P. megistus-borne T. cruzi transmission foci in the vicinity of preserved forest patches in Brasília [On a more local scale, we found a thriving the wild . Althougc forest , 39; in irculate  suggestswellings , 37, 38.T. cruzi transmission from infected primates  to zoo workers including keepers and veterinarians. If unaware of the infection status of the animals they handle, these workers may be at risk of acquiring the infection while drawing or manipulating biological samples, during surgical or dental procedures, or even when performing necropsies on fresh carcasses. Although needles are involved in most of the accidents reported, T. cruzi transmission can also occur through intact mucosae or apparently intact skin, and possibly via droplets or aerosols [Finally, our results highlight the latent risk of accidental aerosols . ZookeepT. cruzi transmission within a small-primate unit at Brasília zoo. We suspect that the vectors, and possibly also the parasites, originally came from nearby gallery forest – a hypothesis that can be tested with samples from both habitats and high-resolution molecular markers [T. cruzi infections should become routine practice in captive-animal facilities located near known or suspected vector habitat. Pyrethroid insecticide spraying yields efficient, short-term infestation control; in the long run, the use of bug-refractory animal lodgings – i.e., with fewer potential bug-hiding sites  and easier to inspect and treat – could help prevent or slow re-infestation. Early detection and elimination of T. cruzi transmission foci should be of particular interest for captive-breeding and translocation-reintroduction programs involving endangered mammal species. Together with specific training on Chagas disease and T. cruzi-related biohazards, this would also help reduce the risk of accidental T. cruzi transmission from infected mammals or vectors to captive-animal handlers, keepers, and veterinarians.The findings we have presented strongly suggest vector-borne  markers . In prac


Xylella fastidiosa CO33 isolate, retrieved from symptomatic leaves of coffee plant intercepted in northern Italy, is reported. The CO33 genome size is 2,681,926 bp with a GC content of 51.7%.The draft genome sequence of  Xylella fastidiosa is a bacterium that colonizes the xylem vessels of host plants and the mouthparts of its insect vectors. This bacterium causes plant diseases of economic importance in a wide range of host plants (X. fastidiosa genomes sequences are available (http://www.ncbi.nlm.nih.gov/genome/genomes/173?). In 2013, X. fastidiosa was detected in southern Italy, representing the first case of this quarantine bacterium becoming established in Europe  strengthened customs control measures to limit importation of infected plant material. As a result several X. fastidiosa-infected coffee plants (Coffea arabica) originating from Central America were intercepted, mainly in the Netherlands . or of subsp. morus (isolate MUL0034), corroborating the genetic complexity of this plant pathogen bacterium and the role of homologous recombination on X. fastidiosa diversity.The draft genome of 2.73 Mbp . The genLJZW00000000. The version described in this paper is version LJZW00000000.1.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Echinochloa (Poaceae) includes numerous problematic weeds that cause the reduction of crop yield worldwide. To date, DNA sequence information is still limited in the genus Echinochloa. In this study, we completed the entire chloroplast genomes of two Echinochloa species  based on high-throughput sequencing data from their fresh green leaves. The two Echinochloa chloroplast genomes are 139,891 and 139,800 base pairs in length, respectively, and contain 131 protein-coding genes, 79 indels and 466 substitutions helpful for discrimination of the two species. The divergence between the genus Echinochloa and Panicum occurred about 21.6 million years ago, whereas the divergence between E. oryzicola and E. crus-galli chloroplast genes occurred about 3.3 million years ago. The two reported Echinochloa chloroplast genome sequences contribute to better understanding of the diversification of this genus.The genus  Echinochloa (Poaceae) belongs to the subfamily Panicoideae Echinochloa crus-galli (L.) Beauv., which is distributed in paddy or non-paddy fields (such as upland crop areas), is considered as one of the most serious weeds E. crus-galli plants et al. (2010) showed that E. crus-galli plants significantly reduced number of rice tillers and resulted in significant reduction of rice yield during rice-E. crus-galli competition under transplanted rice cultivation Echinochloa oryzicola (Vasing.) Vasing. is common in paddy fields, and also reduces production of rice E. oryzicola is variously treated as E. phyllopogon (Stapf) Kossenko, E. crus-galli var. oryzicola (Vasing.) T. Koyama or a tetraploid E. crus-galli var. oryzicola. E. oryzicola has also been misidentified as E. crus-galli var. formosensis Ohwi E. utilis Ohwi & Yabuno and E. frumentacea Link.) and their wild counterpart using the random amplified polymorphic DNA markers et al. (2002) discriminated E. oryzicola and E. crus-galli by the polymerase chain reaction-restriction fragment length polymorphism analysis trnT-L, trnL-F intergenic spacers, and trnL intron) of chloroplast DNA, a phylogenetic tree grouped 30 accessions belonging to nine species of the genus Echinochloa into five E. crus-galli and E. oryzicola, assuming that Eurasian E. crus-galli (hexaploid) arises from the hybridization between tetraploid E. oryzicola  and an unknown diploid species  Echinochloa.The genus CymbidiumArabidopsis thaliana was investigated based on chloroplast DNA sequences of 77 accessions Jacobaea vulgaris were used for population studies Amaranthus tuberculatus) Coix lacryma-jobiJacobaea vulgarisAsclepias syriaca) Ageratina adenophora) et al. (2010) reported on the complete chloroplast genome sequence of Phoenix dactylifera that was obtained from genomic DNA sequenced by GS FLX et al. (2010) reported the use of 454 sequencing technology to obtain the chloroplast genome sequence of mungbean Compared with the nuclear genome, the chloroplast genome has distinct features, e.g., haploid, maternal inheritance, and high conservation in gene content and genome structure Echinochloa are very important to control effectively these weeds. However, morphology-based classification is difficult for this genus because of diverse taxonomic opinions. Meanwhile, molecular systematic research is constrained by the limited chloroplast DNA sequence information of the genus Echinochloa. To provide more DNA sequence information and insights into evolution of the genus Echinochloa, we employed the new approach to construct the complete chloroplast genome sequences of two Echinochloa species  in the current study. Furthermore, we investigated the phylogenetic divergence time within the Echinochloa genus and among several closely related genera.Species identification and clear understanding of genetic relationship of Echinochloa plant samples (matured spikelet) were collected from two different fields  in September, 2011, from Yuhang County, Hangzhou, Zhejiang Province in China . No specific permissions were required for the collection sites, and the study did not involve endangered or protected species. Collected seeds were used to germinate in the next year after field collection. Five plants of each accession were grown in 5L plastic pots , filled with a 3∶1∶1 mixture of soil: peat: sand in a greenhouse at 28°C/25°C (day/night) with a 16 h photoperiod. Seed weights of each Echinochloa accession were determined by random samples of 100 full matured spikelets from five plants in each of the three replicates, and were compared by t-test. Based on morphological observation, STB03 and BTS02, which had typical morphological features of E. oryzicola and E. crus-galli, respectively, were selected for further study.Five (STB01-05) and three (BTS01-03) Mitotic chromosome numbers were determined through conventional acetocarmine method trnT-L-F region  in the chloroplast genome of STB03 (E. oryzicola) and BTS02  were amplified by PCR with primers CATTACAAATGCGATGCTCT and ATTTGAACTGGTGACACGAG. The sequences were then aligned to those of 30 Echinochloa accessions http://mafft.cbrc.jp/alignment/server/) http://www.megasoftware.net/) DNA was extracted from green leaves of STB03 and BTS02 using routine protocol (CTAB) For chloroplast genome sequencing, total DNA was extracted from green leaves of STB03 and BTS02. DNA was fragmented into 300–400 bp pieces. Libraries with 230 bp insertion size for Illumina Hiseq2000 sequencing platform were constructed according to the manufacturer’s instructions (Illumina). Twenty-five and 50 Gb genome data were obtained for STB03 and BTS02, respectively. Raw data was removed adaptors and qualified with Fastx-toolkit with Q30 and above 50 bp.http://bowtie-bio.sourceforge.net/index.shtml) Panicum virgatum  as reference De novo assembly of the mapped reads was then performed using CLC software (http://www.clcbio.com) with default settings . The gaps were closed by GapCloser (http://soap.genomics.org.cn/soapdenovo.html) P. virgatum) chloroplast genomes and those between STB03 and BTS02 were verified through PCR amplification. All PCR products were sequenced by the Sanger method Echinochloa chloroplast genomes using DOGMA (http://dogma.ccbb.utexas.edu/) http://wolfe.gen.tcd.ie/GenomeVx/) Echinochloa species were deposited into GenBank with accession numbers KJ000048 (STB03) and KJ000047 (BTS02), respectively.Bowtie2  with the default settings (sliding window size 100 bp and minimum width of a conserved region 100 bp) The complete chloroplast genomes of O. sativa, S. bicolor, Zea mays, P. virgatum, E. crus-galli and E. oryzicola) were involved in the analysis on divergence time estimation. Multiple sequence alignments were conducted for all chloroplast genome sequences with MAFFT http://beast.bio.ed.ac.uk/Main_Page) with O. sativa as an outgroup Z. mays-S. bicolor split time  Six species (Echinochloa collection was first identified. In the five (STB01-05) and three (BTS01-03) Echinochloa plants collected in paddy and upland crop fields, respectively, STB03 had a compact plant type, bigger seeds (0.430 g per hundred spikelets), and high similarity with rice (similar plant architecture and leaf color at the seedling stage), which are typical morphological features of E. oryzicola. BTS02 had geniculate culms, smaller seeds , and a loose plant type, which are typical morphological features of E. crus-galliE. oryzicolaE. crus-galli  E. oryzicola and BTS02 with E. crus-galli.For our genome sequencing effort, species of our us-galli . To furtE. oryzicola (STB03) chloroplast-related reads and de novo assembly of these reads. Four gaps (each with size of <20 bp) within the three scaffolds and two inter-scaffold gaps  were closed by PCR amplification coupled with Sanger sequencing. After alignment of the STB03 assembly with the reference (P. virgatum chloroplast genome), the STB03 genome structure  could be determined, showing that only one IR (IRa) was successfully assembled. PCR primers were then designed for the joining of IRb-LSC and IRb-SSC and the validation of two indels between STB03 and the reference. Finally, all clean reads generated by our high-throughput sequencing from STB03 were mapped back to the STB03 assembly. No SNPs were found, suggesting no variations between the two IR regions and also a high quality of our assembly. For the assembly of E. crus-galli (BTS02), the chloroplast genome of E. oryzicola (STB03) was used as the reference and four pairs of primers were designed for gap closure and joining regions of IR-LSC and IR-SSC. Meanwhile, PCR amplification followed by Sanger sequencing was used to validate eight regions with variations between E. oryzicola (STB03) and E. crus-galli (BTS02). Finally, we obtained complete chloroplast genomes of E. oryzicola and E. crus-galli with length of 139,891 and 139,800 bp, respectively.Three scaffolds were generated after collecting The approach by whole-genome high-throughput sequencing without purification of the chloroplast DNA provides a new way to obtain the chloroplast genome and has been successfully used in several studies E. oryzicola and E. crus-galli chloroplast genomes displayed typical quadripartite structure consisting of a pair of IRs  separated by LSC  and SSC  regions , which is generally similar to other chloroplast genomes atpF, ndhB, ndhA, trnK-UUU, trnT-GGU, trnL-UAA, trnV-UAC, trnI-GAU, and trnA-UGC, whereas ycf3 harbors two introns. Some genes should have become pseudogenes because of the early stop codons identified in their coding sequences, such as rps19, rps16, ycf2, ycf15, orf56 and ycf1. The similar mutations have been observed in the chloroplast genomes of other angiosperm species Echinochloa chloroplast genomes.Both  regions . The EchEchinochloa chloroplast genomes were conserved with 99.5% sequence identity and contained similar genes. However, a total of 79 indels , sequence identity was plotted using mVISTA E. oryzicola as the reference  encoding two large open reading frames were frequently lost or became pseudogenes in Poaceae chloroplast genomes rps16 also became a pseudogene in O. sativa, while rps19 was only found as a pseudogene in the Echinochloa chloroplast genomes. Additionally, ycf15 and orf56 were lost or became pseudogenes in any of the species examined.Compared to other Poaceae members and bidopsis , seven gEchinochloa species, chloroplast genomes from six members of the grass family  were used. O. sativa was selected as an outgroup and T. aestivum, the member of the subfamily Pooideae, was excluded in this analysis. The constructed tree based on the whole complete chloroplast genomes shows that the Echinochloa species were first grouped with P. virgatum and then S. bicolor and Z. mays (Echinochloa branched off from the genus Panicum around 21.6 million years ago (Mya), and the divergence date between E. oryzicola and E. crus-galli was around 3.3 Mya. Meanwhile, we estimated the divergence times based on coding sequences of single copy genes, which are slightly younger than those estimated by the whole chloroplast genomes  and an unknown diploid species  E. oryzicola and the unknown diploid species. To determine the speciation time of E. crus-galli, i.e., the hybridization event of E. oryzicola and the unknown diploid maternal parent of E. crus-galli, nuclear DNA sequences are necessary.The subfamily Panicoideae (Poaceae) is comprised of two major tribes, i.e., Paniceae that includes Echinochloa species, providing more DNA sequence information for genetic relationship and population studies on this genus. Furthermore, the phylogenetic divergence time estimated based on the chloroplast genome sequences could be useful to better understand the evolution of the genus Echinochloa.In summary, we obtained the entire chloroplast genomes of two Figure S1The first 20%  and 10%  MCMC samples have been discarded as burn in.(PPT)Click here for additional data file.Figure S2The images of STB03and BTS02 show that the former has some typical morphological traits of E. oryzicola, such as a compact plant type, bigger seeds, and high similarity with rice, whereas the latter has traits of E. crus-galli, such as geniculate culms, smaller seeds, and a loose type.(PPT)Click here for additional data file.Figure S3Chromosome numbers show that STB03 (2n = 4x = 36) is tetraploid (A) and BTS02 (2n = 6x = 54) is hexaploid (B). The numbers were determined by the conventional acetocarmine method.(PPT)Click here for additional data file.Figure S4Phylogenetic relationships among STB03, BTS02, and 30 Echinochloa accessions based on the nucleotide sequences of the trnT-L-F region of the chloroplast genome. The tree was constructed using the ML method. Bootstrap values with less than 50 are not shown.(PPT)Click here for additional data file.Figure S5The E. crus-galli chloroplast genome structure and annotation. Outer circle: The genes shown outside of the circle are transcribed clockwise, whereas those inside are transcribed counterclockwise; Inner circle: the genomic structure with two inverted repeats (IR) and two single copy regions (LSC and SSC). Genes belonging to different functional groups are color coded.(PPT)Click here for additional data file.Figure S6Divergence time of the genus Echinochloa. Divergence time was estimated using BEAST based on coding sequences of single copy genes shared among the six species . The numbers showed at nodes indicate divergence time.(PPT)Click here for additional data file.Table S1Primers used in the assembly of two Echinochloa chloroplast genomes.(XLS)Click here for additional data file.Table S2The performed MCMC runs, burn in and the effective sample size (ESS).(XLS)Click here for additional data file.Table S3Indels found in the Echinochloa chloroplast genomes.(XLS)Click here for additional data file.Table S4Number of protein-coding genes in the seven chloroplast genomes .(XLS)Click here for additional data file.