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Sökning: WFRF:(Baldauf Sandra L)

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1.
  • Cavender, James C., et al. (författare)
  • New dictyostelid cellular slime molds from South Africa
  • 2018
  • Ingår i: Phytotaxa. - : MAGNOLIA PRESS. - 1179-3155 .- 1179-3163. ; 383:3, s. 233-251
  • Tidskriftsartikel (refereegranskat)abstract
    • A distributional study of the dictyostelid cellular slime molds (dictyostelids) of South Africa was carried out during 2006 as part of the Global Biodiversity of Eumycetozoans project based at the University of Arkansas and funded by the National Science Foundation Samples of soil/humus collected from 31 study sites yielded a total of 881 clones, with an average density of 210 clones/gram for all samples in which dictyostelids were detected. Eighteen different species were represented by one or more clones, and six of these could not be identified. These six species are described herein as new to science. In addition, information is provided on the ecological distribution of all of the species of dictyostelids now known to occur in South Africa.
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4.
  • Perrigo, Allison L., et al. (författare)
  • What's on your boots : an investigation into the role we play in protist dispersal
  • 2012
  • Ingår i: Journal of Biogeography. - : Wiley. - 0305-0270 .- 1365-2699. ; 39:5, s. 998-1003
  • Tidskriftsartikel (refereegranskat)abstract
    • D. M. Wilkinson (2010, Journal of Biogeography, 37, 393–397) suggested that anthropogenic dispersal is an understudied and potentially important factor in terrestrial protist biogeography. We investigated human footwear as a potential vector of dictyostelids, a diverse group of amoebae that includes both geographically restricted and cosmopolitan species. Eighteen pairs of boots were examined and dictyostelids were isolated from nearly all samples larger than 5.0 g. In total, six dictyostelid isolates were recovered, corresponding to four species –Dictyostelium minutum, D. sphaerocephalum, D. leptosomopsis and a new species, Polysphondylium sp. 1. Myxogastrid amoebae and acrasid-like aggregations were also observed. Thus anthropogenic dispersal of naked amoebae appears to occur. The possible role of variations in dictyostelid fruiting body morphologies in dispersal potential is also discussed. These results support Wilkinson’s proposal and suggest that dictyostelids may be a useful group with which to study anthropogenic dispersal of terrestrial protists.
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5.
  • Romeralo, Maria, et al. (författare)
  • An expanded phylogeny of social amoebas (Dictyostelia) shows increasing diversity and new morphological patterns
  • 2011
  • Ingår i: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 11, s. 84-
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Social Amoebae or Dictyostelia are eukaryotic microbes with a unique life cycle consisting of both uni- and multicellular stages. They have long fascinated molecular, developmental and evolutionary biologists, and Dictyostelium discoideum is now one of the most widely studied eukaryotic microbial models. The first molecular phylogeny of Dictyostelia included most of the species known at the time and suggested an extremely deep taxon with a molecular depth roughly equivalent to Metazoa. The group was also shown to consist of four major clades, none of which correspond to traditional genera. Potential morphological justification was identified for three of the four major groups, on the basis of which tentative names were assigned. Results: Over the past four years, the Mycetozoan Global Biodiversity Survey has identified many new isolates that appear to be new species of Dictyostelia, along with numerous isolates of previously described species. We have determined 18S ribosomal RNA gene sequences for all of these new isolates. Phylogenetic analyses of these data show at least 50 new species, and these arise from throughout the dictyostelid tree breaking up many previously isolated long branches. The resulting tree now shows eight well-supported major groups instead of the original four. The new species also expand the known morphological diversity of the previously established four major groups, violating nearly all previously suggested deep morphological patterns. Conclusions: A greatly expanded phylogeny of Dictyostelia now shows even greater morphological plasticity at deep taxonomic levels. In fact, there now seem to be no obvious deep evolutionary trends across the group. However at a finer level, patterns in morphological character evolution are beginning to emerge. These results also suggest that there is a far greater diversity of Dictyostelia yet to be discovered, including novel morphologies.
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6.
  • Sheikh, Sanea, et al. (författare)
  • A New Classification of the Dictyostelids
  • 2018
  • Ingår i: Protist. - : Elsevier BV. - 1434-4610 .- 1618-0941. ; 169:1, s. 1-28
  • Tidskriftsartikel (refereegranskat)abstract
    • Traditional morphology-based taxonomy of dictyostelids is rejected by molecular phylogeny. A new classification is presented based on monophyletic entities with consistent and strong molecular phylogenetic support and that are, as far as possible, morphologically recognizable. All newly named clades are diagnosed with small subunit ribosomal RNA (18S rRNA) sequence signatures plus morphological synapomorphies where possible. The two major molecular clades are given the rank of order, as Acytosteliales ord. nov. and Dictyosteliales. The two major clades within each of these orders are recognized and given the rank of family as, respectively, Acytosteliaceae and Cavenderiaceae fam. nov. in Acytosteliales, and Dictyosteliaceae and Raperosteliaceae fam. nov. in Dictyosteliales. Twelve genera are recognized: Cavenderia gen. nov. in Cavenderiaceae, Acytostelium, Rostrostelium gen. nov. and Heterostelium gen. nov. in Acytosteliaceae, Tieghemostelium gen. nov., Hagiwaraea gen. nov., Raperostelium gen. nov. and Speleostelium gen. nov. in Raperosteliaceae, and Dictyostelium and Polysphondylium in Dictyosteliaceae. The “polycephalum” complex is treated as Coremiostelium gen. nov. (not assigned to family) and the “polycarpum” complex as Synstelium gen. nov. (not assigned to order and family). Coenonia, which may not be a dictyostelid, is treated as a genus incertae sedis. Eighty-eight new combinations are made at species and variety level, and Dictyostelium ammophilum is validated.
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7.
  • Zhao, Weizhou (författare)
  • Evolution of streamlined genomes in ultra-small aquatic bacteria
  • 2019
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • This thesis investigates the evolutionary processes of streamlined genomes from aquatic bacteria adapting to different salinities, using two groups of ultra-small aquatic bacteria (LD12 Alphaproteobacteria and acI Actinobacteria). Due to difficulties in obtaining pure cultures of these bacteria, culture-free approaches (single-cell genomics and metagenomics) were used to construct and compare genomes, and to study the mechanisms and selective forces of adaptation to freshwater, brackish, and marine ecosystems.A study of single-cell amplified genomes (SAGs) from freshwater LD12 Alphaproteobacteria revealed that LD12 forms a clade embedded within the globally dominant marine Alphaproteobacteria SAR11, and subclades were organized into distinct microclusters. LD12 genomes had a very low ratio of recombination to point mutations, in contrast to their marine relatives which had a very high ratio of recombination to mutation. We suggested that the transition from marine to freshwater was a bottleneck event, resulting in reduced opportunities for recombination.In a separate study, we analyzed complete genomes and SAGs from acI Actinobacteria abundant in freshwater ecosystems, and found overall low rates of sequence divergence with however a dramatic acceleration near genomic island 1 (GI-1). We also identified a type IV topoisomerase, the delta subunit of DNA polymerase, and an RNA polymerase sigma factor near GI-1. Based on these results, we proposed a model for the evolution and expression of novel genes in these genomes.We also isolated and analyzed the genomes of single cells from a marine Actinobacteria (subclass Candidatus Actinomarinidae). These were not related to acI, but to Acidimicrobiia, which suggested salinity barriers have been crossed several times by Actinobacteria.To further understand the transition to different salinities, we obtained acI SAGs from three different intermediate-salinity Baltic Sea locations. We took sequence reads from 21 metagenomes taken along the salinity gradient, and recruited these fragments to both the freshwater and brackish acI reference genomes. These results indicated that transitions between fresh and brackish waters have occurred multiple times in acI Actinobacteria and some of these strains are globally present in coastal waters.
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8.
  • Ajawatanawong, Pravech, et al. (författare)
  • Evolution of protein indels in plants, animals and fungi
  • 2013
  • Ingår i: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 13, s. 140-
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Insertions/deletions (indels) in protein sequences are useful as drug targets, protein structure predictors, species diagnostics and evolutionary markers. However there is limited understanding of indel evolutionary patterns. We sought to characterize indel patterns focusing first on the major groups of multicellular eukaryotes. Results: Comparisons of complete proteomes from a taxonically broad set of primarily Metazoa, Fungi and Viridiplantae yielded 299 substantial (>250aa) universal, single-copy (in-paralog only) proteins, from which 901 simple (present/absent) and 3,806 complex (multistate) indels were extracted. Simple indels are mostly small (1-7aa) with a most frequent size class of 1aa. However, even these simple looking indels show a surprisingly high level of hidden homoplasy (multiple independent origins). Among the apparently homoplasy-free simple indels, we identify 69 potential clade-defining indels (CDIs) that may warrant closer examination. CDIs show a very uneven taxonomic distribution among Viridiplante (13 CDIs), Fungi (40 CDIs), and Metazoa (0 CDIs). An examination of singleton indels shows an excess of insertions over deletions in nearly all examined taxa. This excess averages 2.31 overall, with a maximum observed value of 7.5 fold. Conclusions: We find considerable potential for identifying taxon-marker indels using an automated pipeline. However, it appears that simple indels in universal proteins are too rare and homoplasy-rich to be used for pure indel-based phylogeny. The excess of insertions over deletions seen in nearly every genome and major group examined maybe useful in defining more realistic gap penalties for sequence alignment. This bias also suggests that insertions in highly conserved proteins experience less purifying selection than do deletions.
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9.
  • Ajawatanawong, Pravech, et al. (författare)
  • SeqFIRE : a web application for automated extraction of indel regions and conserved blocks from protein multiple sequence alignments
  • 2012
  • Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:W1, s. W340-W347
  • Tidskriftsartikel (refereegranskat)abstract
    • Analyses of multiple sequence alignments generally focus on well-defined conserved sequence blocks, while the rest of the alignment is largely ignored or discarded. This is especially true in phylogenomics, where large multigene datasets are produced through automated pipelines. However, some of the most powerful phylogenetic markers have been found in the variable length regions of multiple alignments, particularly insertions/deletions (indels) in protein sequences. We have developed Sequence Feature and Indel Region Extractor (SeqFIRE) to enable the automated identification and extraction of indels from protein sequence alignments. The program can also extract conserved blocks and identify fast evolving sites using a combination of conservation and entropy. All major variables can be adjusted by the user, allowing them to identify the sets of variables most suited to a particular analysis or dataset. Thus, all major tasks in preparing an alignment for further analysis are combined in a single flexible and user-friendly program. The output includes a numbered list of indels, alignments in NEXUS format with indels annotated or removed and indel-only matrices. SeqFIRE is a user-friendly web application, freely available online at www.seqfire.org/.
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10.
  • Al Jewari, Caesar, et al. (författare)
  • An excavate root for the eukaryote tree of life
  • 2023
  • Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:17
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
    • Much of the higher-order phylogeny of eukaryotes is well resolved, but the root remains elusive. We assembled a dataset of 183 eukaryotic proteins of archaeal ancestry to test this root. The resulting phylogeny identifies four lineages of eukaryotes currently classified as “Excavata” branching separately at the base of the tree. Thus, Parabasalia appear as the first major branch of eukaryotes followed sequentially by Fornicata, Preaxostyla, and Discoba. All four excavate branch points receive full statistical support from analyses with commonly used evolutionary models, a protein structure partition model that we introduce here, and various controls for deep phylogeny artifacts. The absence of aerobic mitochondria in Parabasalia, Fornicata, and Preaxostyla suggests that modern eukaryotes arose under anoxic conditions, probably much earlier than expected, and without the benefit of mitochondrial respiration.
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11.
  • Al Jewari, Caesar, et al. (författare)
  • Conflict over the Eukaryote Root Resides in Strong Outliers, Mosaics and Missing Data Sensitivity of Site-Specific (CAT) Mixture Models
  • 2022
  • Ingår i: Systematic Biology. - : Oxford University Press (OUP). - 1063-5157 .- 1076-836X.
  • Tidskriftsartikel (refereegranskat)abstract
    • Phylogenetic reconstruction using concatenated loci ("phylogenomics" or "supermatrix phylogeny") is a powerful tool for solving evolutionary splits that are poorly resolved in single gene/protein trees. However, recent phylogenomic attempts to resolve the eukaryote root have yielded conflicting results, along with claims of various artifacts hidden in the data. We have investigated these conflicts using two new methods for assessing phylogenetic conflict. ConJak uses whole marker (gene or protein) jackknifing to assess deviation from a central mean for each individual sequence, whereas ConWin uses a sliding window to screen for incongruent protein fragments (mosaics). Both methods allow selective masking of individual sequences or sequence fragments in order to minimize missing data, an important consideration for resolving deep splits with limited data. Analyses focused on a set of 76 eukaryotic proteins of bacterial ancestry previously used in various combinations to assess the branching order among the three major divisions of eukaryotes: Amorphea (mainly animals, fungi, and Amoebozoa), Diaphoretickes (most other well-known eukaryotes and nearly all algae) and Excavata, represented here by Discoba (Jakobida, Heterolobosea, and Euglenozoa). ConJak analyses found strong outliers to be concentrated in undersampled lineages, whereas ConWin analyses of Discoba, the most undersampled of the major lineages, detected potentially incongruent fragments scattered throughout. Phylogenetic analyses of the full data using an LG-gamma model support a Discoba sister scenario (neozoan-excavate root), which rises to 99-100% bootstrap support with data masked according to either protocol. However, analyses with two site-specific (CAT) mixture models yielded widely inconsistent results and a striking sensitivity to missing data. The neozoan-excavate root places Amorphea and Diaphoretickes as more closely related to each other than either is to Discoba, a fundamental relationship that should remain unaffected by additional taxa.
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  • Al Jewari, Caesar (författare)
  • Resolving deep nodes of eukaryote phylogeny
  • 2022
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • My thesis aims to solve deep nodes in the eukaryote tree of life (eToL), by developing new data sets and new approaches to analysing them. In paper I, I described a dataset of 76 universal eukaryotic proteins of bacterial descent (euBacs), in order to test the relations among the three main divisions of mitochondriate eukaryotes (Amorphea, Diaphretickes and Discoba). I developed two protocols to identify problematic data. The conJac protocol analyzes data by jackknifing to detect outlier sequences, while conWin uses a sliding window to find sequence fragments of potentially foreign origin. Phylogenetic analyses of the 76 euBacs, with and without conWin or conJac filtering place Discoba as the sister group to Amorphea and Diaphretickes. The results are largely consistent and highly supported under various evolutionary models except for highly complex CAT models. In paper II, I describe a dataset of 198 universal eukaryote proteins of archaeal ancestry (euArcs), which includes the remaining eukaryotes, informally referred to as amitochondriate excavate. These were excluded from the previous study because they lack euBacs. Phylogenetic analyses of the euArc dataset place the amitochondriate excavate as the first three branches of eToL, followed by Discoba, the only mitochondriate excavates, which appear as a sister group to the remaining eukaryotes. I also developed a protocol using predicted protein structures to increase the fitness of the model without inflating the parameter space, allowing me to conduct a series of control analyses and further support the multi-excavate root. In Paper III, I describe a new application of reciprocal-rooting using concatenated sequences, which I then use to test the euArc root. I also developed two sampling protocols unique to this kind of data. The protocols confirm the multi-excavate euArc root, which indicates that eukaryotes arose from an excavate ancestor. Paper IV describes a follow-up on the ConWin results from Paper I. These show moderate to strong support for mosaicism in 16 euBac proteins from diverse metabolic pathways and donor lineages. In summary, this thesis presents a novel root for the eukaryote tree of life. The new root requires revision of fundamental theories of eukaryote evolution including the source and timing of mitochondrial origins. The methods I have developed are applicable to many different kinds of phylogenetic studies, and the new protein structure model should make these analyses faster, more flexible, and more widely available.
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14.
  • Atkinson, Gemma C., et al. (författare)
  • Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components
  • 2008
  • Ingår i: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 8, s. 290-
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Members of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment and phylogenetic analysis. Results: Extensive BLAST searches confirm that Hbs1p and eRF3 are limited to eukaryotes, while Dom34p and eRF1 (a/eRF1) are universal in eukaryotes and archaea. Ski7p appears to be restricted to a subset of Saccharomyces species. Alignments show that Dom34p does not possess the characteristic class-1 RF minidomains GGQ, NIKS and YXCXXXF, in line with recent crystallographic analysis of Dom34p. Phylogenetic trees of the protein families allow us to reconstruct the evolution of mRNA surveillance mechanisms mediated by these proteins in eukaryotes and archaea. Conclusion: We propose that the last common ancestor of eukaryotes and archaea possessed Dom34p-mediated no-go decay (NGD). This ancestral Dom34p may or may not have required a trGTPase, mostly like a/eEF1A, for its delivery to the ribosome. At an early stage in eukaryotic evolution, eEF1A was duplicated, giving rise to eRF3, which was recruited for translation termination, interacting with eRF1. eRF3 evolved nonsense-mediated decay (NMD) activity either before or after it was again duplicated, giving rise to Hbs1p, which we propose was recruited to assist eDom34p in eukaryotic NGD. Finally, a third duplication within ascomycete yeast gave rise to Ski7p, which may have become specialised for a subset of existing Hbs1p functions in non-stop decay (NSD). We suggest Ski7p-mediated NSD may be a specialised mechanism for counteracting the effects of increased stop codon read-through caused by prion-domain [ PSI+] mediated eRF3 precipitation.
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15.
  • Baldauf, Sandra L., et al. (författare)
  • A Deep Hidden Diversity of Dictyostelia
  • 2018
  • Ingår i: Protist. - : Elsevier BV. - 1434-4610 .- 1618-0941. ; 169:1, s. 64-78
  • Tidskriftsartikel (refereegranskat)abstract
    • Dictyostelia is a monophyletic group of transiently multicellular (sorocarpic) amoebae, whose study is currently limited to laboratory culture. This tends to favour faster growing species with robust sorocarps, while species with smaller more delicate sorocarps constitute most of the group’s taxonomic breadth. The number of known species is also small (∼150) given Dictyostelia’s molecular depth and apparent antiquity (>600 myr). Nonetheless, dictyostelid sequences are rarely recovered in culture independent sampling (ciPCR) surveys. We developed ciPCR primers to specifically target dictyostelid small subunit (SSU or 18S) rDNA and tested them on total DNAs extracted from a wide range of soils from five continents. The resulting clone libraries show mostly dictyostelid sequences (∼90%), and phylogenetic analyses of these sequences indicate novel lineages in all four dictyostelid families and most genera. This is especially true for the species-rich Heterostelium and Dictyosteliaceae but also the less species-rich Raperosteliaceae. However, the most novel deep branches are found in two very species-poor taxa, including the deepest branch yet seen in the highly divergent Cavenderiaceae. These results confirm a deep hidden diversity of Dictyostelia, potentially including novel morphologies and developmental schemes. The primers and protocols presented here should also enable more comprehensive studies of dictyostelid ecology.
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16.
  • Baldauf, Sandra L. (författare)
  • An overview of the phylogeny and diversity of eukaryotes
  • 2008
  • Ingår i: Journal of systematics and evolution. - 1674-4918. ; 46:3, s. 263-273
  • Tidskriftsartikel (refereegranskat)abstract
    • Our understanding of eukaryote biology is dominated by the study of land plants, animals and fungi. However, these are only three isolated fragments of the full diversity of extant eukaryotes. The majority of eukaryotes, in terms of major taxa and probably also sheer numbers of cells, consists of exclusively or predominantly unicellular lineages. A surprising number of these lineages are poorly characterized. Nonetheless, they are fundamental to our understanding of eukaryote biology and the underlying forces that shaped it. This article consists of an overview of the current state of our understanding of the eukaryote tree. This includes the identity of the major groups of eukaryotes, some of their important, defining or simply interesting features and the proposed relationships of these groups to each other.
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  • Carr, Martin, et al. (författare)
  • Conserved Meiotic Genes Point to Sex in the Choanoflagellates
  • 2010
  • Ingår i: Journal of Eukaryotic Microbiology. - : Wiley. - 1066-5234 .- 1550-7408. ; 57:1, s. 56-62
  • Tidskriftsartikel (refereegranskat)abstract
    • The choanoflagellates are a widespread group of heterotrophic aquatic nanoflagellates, which have recently been confirmed as the sister-group to Metazoa. Asexual reproduction is the only mode of cell division that has been observed within the group; at present the range of reproductive modes, as well as the ploidy level, within choanoflagellates are unknown. The recent discovery of long terminal repeat retrotransposons within the genome of Monosiga brevicollis suggests that this species also has sexual stages in its life cycle because asexual organisms cannot tolerate retrotransposons due to the rapid accumulation of deleterious mutations caused by their transposition. We screened the M. brevicollis genome for known eukaryotic meiotic genes, using a recently established "meiosis detection toolkit" of 19 genes. Eighteen of these genes were identified, none of which appears to be a pseudogene. Four of the genes were also identified in expressed sequence tag data from the distantly related Monosiga ovata. The presence of these meiosis-specific genes provides evidence for meiosis, and by implication sex, within this important group of protists.
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19.
  • Carr, M., et al. (författare)
  • Molecular phylogeny of choanoflagellates, the sister group to Metazoa
  • 2008
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:43, s. 16641-16646
  • Tidskriftsartikel (refereegranskat)abstract
    • Choanoflagellates are single-celled aquatic flagellates with a unique morphology consisting of a cell with a single flagellum surrounded by a "collar" of microvilli. They have long interested evolutionary biologists because of their striking resemblance to the collared cells (choanocytes) of sponges. Molecular phylogeny has confirmed a close relationship between choanoflagellates and Metazoa, and the first choanoflagellate genome sequence has recently been published. However, molecular phylogenetic studies within choanoflagellates are still extremely limited. Thus, little is known about choanoflagellate evolution or the exact nature of the relationship between choanoflagellates and Metazoa. We have sequenced four genes from a broad sampling of the morphological diversity of choanoflagellates including most species currently available in culture. Phylogenetic analyses of these sequences, alone and in combination, reject much of the traditional taxonomy of the group. The molecular data also strongly support choanoflagellate monophyly rejecting proposals that Metazoa were derived from a true choanoflagellate ancestor. Mapping of a complementary matrix of morphological and ecological traits onto the phylogeny allows a reinterpretation of choanoflagellate character evolution and predicts the nature of their last common ancestor.
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20.
  • Davis, Robert B, et al. (författare)
  • Many hexapod groups originated earlier and withstood extinction events better than previously realized : inferences from supertrees
  • 2010
  • Ingår i: Proceedings of the Royal Society of London. Biological Sciences. - : The Royal Society. - 0962-8452 .- 1471-2954. ; 277:1687, s. 1597-1606
  • Tidskriftsartikel (refereegranskat)abstract
    • Comprising over half of all described species, the hexapods are central to understanding the evolution of global biodiversity. Direct fossil evidence suggests that new hexapod orders continued to originate from the Jurassic onwards, and diversity is presently higher than ever. Previous studies also suggest that several shifts in net diversification rate have occurred at higher taxonomic levels. However, their inferred timing is phylogeny dependent. We re-examine these issues using the supertree approach to provide, to our knowledge, the first composite estimates of hexapod order-level phylogeny. The Purvis matrix representation with parsimony method provides the most optimal supertree, but alternative methods are considered. Inferring ghost ranges shows richness of terminal lineages in the order-level phylogeny to peak just before the end-Permian extinction, rather than the present day, indicating that at least 11 more lineages survived this extinction than implied by fossils alone. The major upshift in diversification is associated with the origin of wings/wing folding and for the first time, to our knowledge, significant downshifts are shown associated with the origin of species-poor taxa (e.g. Neuropterida, Zoraptera). Polyneopteran phylogeny, especially the position of Zoraptera, remains important resolve because this influences findings regarding shifts in diversification. Our study shows how combining fossil with phylogenetic information can improve macroevolutionary inferences.
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21.
  • Elmhalli, Fawzeia, PhD, et al. (författare)
  • Acaricidal activity against Ixodes ricinus nymphs of essential oils from the Libyan plants Artemisia herba alba, Origanum majorana and Juniperus phoenicea
  • 2021
  • Ingår i: Veterinary Parasitology. - : Elsevier BV. - 2405-9390. ; 24
  • Tidskriftsartikel (refereegranskat)abstract
    • Ixodes ricinus (L.) (Acari: Ixodidae) is a major vector for the transmission of several important human pathogens. The aim of the present study was to evaluate the in vitro efficacy of different concentrations of essential oils (Eos) on I. ricinus tick nymphs. Oils were obtained from the leaves of three plants native to Libya: white wormwood (Artemisia herba alba Asso), marjoram (Origanum majorana L.) and Arar (Juniperus phoenicea L., English common name Phoenician juniper). Assays were done using the "open filter paper method". Two concentrations from each oil, 0.5 and 1 mu l/cm, were tested. The acaricidal effect was measured in terms of the lethal concentrations (LC50, LC95) and lethal time (LT50, LT95). Mortality rates were obtained by counting the surviving nymphs every 30 min for the first five hours and then at 24, 48 and 72 h. A mortality of 100% was recorded at the higher concentration of oils (1 mu l/cm(2)) from A. herba alba and J. phoenicea at the first 2 h of exposure. Exposure to O. majorana led to 100% mortality on the third day (72 h), and this effect decreased noticeably with 0.5 mu l/cm(2) oil at the same exposure time. However, 50% of ticks showed a paralysis effect and less movement after 2 h. The LC50 of mortality was reached within the first 24 h of exposure time at 0.5 mu l/cm(2) of O. majorana, which produced 60% tick's mortality. Chemical composition of the essential oils was elucidated by gas chromatography-mass spectrometry analyses. These results suggest that essential oils deserve further investigation as components of alternative approaches for I. ricinus tick control.
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22.
  • Elmhalli, Fawzeia, PhD, et al. (författare)
  • The repellency and toxicity effects of Essential oils from the Libyan plants Salvadora persica and Rosmarinus officinalis against nymphs of Ixodes ricinus.
  • 2019
  • Ingår i: Experimental & applied acarology. - : Springer Science and Business Media LLC. - 0168-8162 .- 1572-9702. ; 77:4, s. 585-599
  • Tidskriftsartikel (refereegranskat)abstract
    • Essential oils extracted from the leaves of Libyan Rosemary (Rosmarinus officinalis L.), and Miswak (Salvadora persica L.) were evaluated for their acaricidal and repellent effects on Ixodes ricinus L. nymphs (Acari: Ixodidae) using a bioassay based on an “open filter paper method".  R. officinalis leaf essential oil diluted to 0.5 and 1µl/cm ² in acetone exhibited, respectively, 20% and 100% tick mortality after about 5 hours of exposure. A total of 50% and 95% of I. ricinus nymphs were killed by direct contact with the oil when exposed to lethal concentrations (LC) of 0.7µl/cm² (LC50) and 0.95 µl/cm² (LC95), respectively. The LC50 (0.5µl/cm²) was reached before the end of the first 24 hours of exposure time (ET), as tick mortality at 24 hours was 60%. S. persica leaf essential oil at 1µl/cm² showed a significant repellency effect against I. ricinus nymphs at 1.5 hours ET. A 95% repellency was observed at a repellent concentration (RC95) of 1µl/cm² of S. persica, but no significant mortality was recorded at this dose of S. persica oil. Gas chromatography-mass spectrometry analyses showed that the main monoterpenes in both oils were 1,8-cineol, α-pinene, and β-pinene, although in markedly different proportions. These results suggest that essential oils have substantial potential as alternative approaches for I. ricinus tick control.
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23.
  • Elmhalli, Fawzeia, et al. (författare)
  • Toxicity against Ixodes ricinus nymphs of essential oils from the Libyan plants Artemisia herba alba, Origanum majorana and Juniperus phoenicea
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • ABSTRACT Ixodes ricinus (L.) (Acari: Ixodidae) is a major vector for the transmission of several important human pathogens. The aim of the study was to evaluate the in vitro efficacy of different concentrations of essential oils (Eos) on I. ricinus tick nymphs. Oils were obtained from the leaves of three plants native to Libya: white wormwood (Artemisia herba alba Asso), marjoram (Origanum majorana L.) and Arâr (Juniperus phoenicea L., English common name Phoenician juniper). Assays were done using the “open filter paper method". Two concentrations from each oil, 0.5 and 1µl/cm, were tested. And the toxic effect was measured in terms of the lethal concentrations (LC50, LC95) and lethal time (LT50, LT95). Mortality rates were obtained by counting the surviving nymphs every 30 minutes for the first five hours and then at 24, 48 and 72 h. A mortality of 100% was recorded at the higher concentration of oils (1µl/cm²) from A. herba alba and J. phoenicea at the first 2 hours of exposure. With O. majorana 100% mortality was only reached on the third day (72h), and this effect decreased noticeably with 0.5µl/cm² oil at the same exposure time. However, 50% of ticks showed a paralysis effect and less movement after 2 hours. Whereas the LC50 of mortality was reached within the first 24h of ET at 0.5µl/cm² of O. majorana, which produced 60% tick’s mortality. Chemical composition of the essential oils was elucidated by gas chromatography-mass spectrometry analyses. These results suggest that essential oils merit further investigation as components of alternative approaches for I. ricinus tick control. 
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24.
  • Field, Dawn, et al. (författare)
  • The minimum information about a genome sequence (MIGS) specification.
  • 2008
  • Ingår i: Nature biotechnology. - : Springer Science and Business Media LLC. - 1546-1696 .- 1087-0156. ; 26:5, s. 541-7
  • Tidskriftsartikel (refereegranskat)abstract
    • With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the 'transparency' of the information contained in existing genomic databases.
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25.
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26.
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27.
  • Fu, Cheng-Jie, et al. (författare)
  • Missing Genes, Multiple ORFs, and C-to-U Type RNA Editing in Acrasis kona (Heterolobosea, Excavata) Mitochondrial DNA
  • 2014
  • Ingår i: Genome Biology and Evolution. - : Oxford University Press (OUP). - 1759-6653. ; 6:9, s. 2240-2257
  • Tidskriftsartikel (refereegranskat)abstract
    • Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs(mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida.
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28.
  • Gray, Michael A., et al. (författare)
  • The response of avian feeding guilds to tropical forest disturbance.
  • 2007
  • Ingår i: Conservation Biology. - : Wiley. - 0888-8892 .- 1523-1739. ; 21:1, s. 133-141
  • Tidskriftsartikel (refereegranskat)abstract
    • Anthropogenic habitat disturbance is a major threat to tropical forests and understanding the ecological consequences of this disturbance is crucial for the conservation of biodiversity. There have been many attempts to determine the ecological traits associated with bird species' vulnerability to disturbance, but no attempt has been made to synthesize these studies to show consensus. We analyzed data from 57 published studies (covering 1214 bird species) that investigated the response of tropical bird assemblages to moderate forest disturbance (e.g., selective logging). Our results show that the mean abundance of species from six commonly reported feeding guilds responded differently to disturbance and that species' ecological traits (body size, local population size, and geographic range size) and evolutionary relationships may influence responses in some guilds. Granivore abundance increased significantly and insectivore and frugivore abundance decreased significantly following disturbance. These general conclusions were robust to the effects of ecological traits and phylogeny. Responses of carnivores, nectarivores, and omnivores were less clear, but analyses that accounted for phylogeny indicated that these guilds declined following disturbance. In contrast to the other guilds, the reported responses of carnivores and nectarivores differed among regions (Asia vs. Neotropics) and were influenced by the sampling protocols used in different studies (e.g., time since disturbance), which may explain the difficulty in detecting general responses to disturbance in these guilds. Overall, general patterns governed the responses of species to habitat disturbance, and the differential responses of guilds suggested that disturbance affects trophic organization and thus ecosystem functioning.
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29.
  • He, Ding, et al. (författare)
  • An Alternative Root for the Eukaryote Tree of Life
  • 2014
  • Ingår i: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 24:4, s. 465-470
  • Tidskriftsartikel (refereegranskat)abstract
    • The root of the eukaryote tree of life defines some of the most fundamental relationships among species. It is also critical for defining the last eukaryote common ancestor (LECA), the shared heritage of all extant species. The unikont-bikont root has been the reigning paradigm for eukaryotes for more than 10 years but is becoming increasingly controversial. We developed a carefully vetted data set, consisting of 37 nuclear-encoded proteins of close bacterial ancestry (euBacs) and their closest bacterial relatives, augmented by deep sequencing of the Acrasis kona (Heterolobosea, Discoba) transcriptome. Phylogenetic analysis of these data produces a highly robust, fully resolved global phy- logeny of eukaryotes. The tree sorts all examined eukaryotes into three megagroups and identifies the Discoba, and potentially its parent taxon Excavata, as the sister group to the bulk of known eukaryote diversity, the proposed Neozoa (Amorphea + Stramenopila+Alveolata+Rhizaria+ Plantae [SARP]). All major alternative hypotheses are rejected with as little as w50% of the data, and this resolu- tion is unaffected by the presence of fast-evolving alignment positions or distant outgroup sequences. This ‘‘neozoan- excavate’’ root revises hypotheses of early eukaryote evolution and highlights the importance of the poorly stud- ied Discoba for understanding the evolution of eukaryotic diversity and basic cellular processes. 
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30.
  • Peacock, Christopher S, et al. (författare)
  • Comparative genomic analysis of three Leishmania species that cause diverse human disease.
  • 2007
  • Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 39:7, s. 839-847
  • Tidskriftsartikel (refereegranskat)abstract
    • Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania: Leishmania infantum and Leishmania braziliensis. The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only 200 genes with a differential distribution between the three species. L. braziliensis, contrary to Leishmania species examined so far, possesses components of a putative RNA-mediated interference pathway, telomere-associated transposable elements and spliced leader–associated SLACS retrotransposons. We show that pseudogene formation and gene loss are the principal forces shaping the different genomes. Genes that are differentially distributed between the species encode proteins implicated in host-pathogen interactions and parasite survival in the macrophage.
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31.
  • Perrigo, Allison L (författare)
  • Diversity Underfoot : Systematics and Biogeography of the Dictyostelid Social Amoebae
  • 2013
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Dictyostelids (Amoebozoa) are a group of social amoebae consisting of approximately 150 species, which are found in terrestrial habitats worldwide. They are divided into eight major clades based on molecular phylogeny, and within these clades are many species complexes. Some species are seemingly cosmopolitan in distribution, while others are geographically restricted. In this thesis dictyostelids were recovered from high latitude habitats (soils in Sweden and Iceland) as well as from the soles of shoes. Morphological characters and DNA sequence analyses were used to identify isolates that were recovered and delimit new species, as well as to investigate the monophyly of Dictyostelium aureostipes. Nine species were reported from Northern Sweden and four from Iceland. Among the isolates recorded in Sweden were two new species, described as D. barbibulus and Polysphondylium fuscans. P. fuscans was among the four species recovered from footwear, contributing evidence for anthropogenic transport of dictyostelids. Ecological patterns were assessed using linear regression and generalized linear models. The ecological analyses of dictyostelids recovered from Iceland indicate that these organisms are most frequently found in soils of near-neutral pH, but also exhibit a species richness peak in moderately acidic soils. These analyses indicate that in Iceland dictyostelid species richness decreases with altitude, and in the northern hemisphere the species richness increases with decreasing latitude. A three-region analysis of the D. aureostipes species complex indicated that this species is in fact made up of at least five phylogenetically distinct clades, and in light of this the group is in need of taxonomic revision. These results indicate that the dictyostelid species richness is higher than previously known, especially in high-latitude regions, and that even seemingly well-defined species may harbour cryptic diversity. Presently, species ranges may be expanding via anthropogenic dispersal but despite this, the dictyostelids are found to exhibit biogeographic trends well known from macroorganisms, such as a latitudinal gradient of species richness.
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32.
  • Romeralo, Maria, et al. (författare)
  • A Fully Resolved Phylogeny of the Social Amoebas (Dictyostelia) Based on Combined SSU and ITS rDNA Sequences
  • 2010
  • Ingår i: Protist. - : Elsevier BV. - 1434-4610 .- 1618-0941. ; 161:4, s. 539-548
  • Tidskriftsartikel (refereegranskat)abstract
    • The dictyostelids possess a complex life cycle including aggregative and multicellular stages. They also include one of the most widely studied protistan model organisms, Dictyostelium discoideum. The current molecular phylogeny of dictyostelids is based largely on SSU (18S) rDNA sequences and shows a deep taxon consisting of four major groups, none of which correspond to the three traditional morphologically-defined genera. However, due to the generally slowly evolving nature of SSU rDNA, these data fail to resolve the majority of branches within the four groups. Given the highly morphologically mixed nature of the dictyostelid groups, it is important to resolve relationships within them. We have determined sequences for the internal transcribed spacers (ITS) of rDNA for nearly all species in the original dictyostelid global phylogeny. Phylogenetic analyses of these data, in combination with the previously determined SSUr DNA sequences, confidently resolve nearly all branches in the tree. This now fully resolved phylogeny confirms the utility of ITS for dictyostelid systematics and lays the ground work for further evolutionary study of the group. (c) 2010 Elsevier GmbH. All rights reserved.
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33.
  • Romeralo, Maria, et al. (författare)
  • A new species of cellular slime mold from southern Portugal based on morphology, ITS and SSU sequences
  • 2009
  • Ingår i: Mycologia. - : Informa UK Limited. - 0027-5514 .- 1557-2536. ; 101:2, s. 269-274
  • Tidskriftsartikel (refereegranskat)abstract
    • Sampling soils to look for dictyostelids in southern Portugal we found an isolate that has a morphology that. differed from any previously described species of the group. We sequenced the internally transcribed spacer (ITS) and small subunit (SSU) genes of the nuclear ribosomal RNA and found that both sequences are distinct front all previously described sequences. Phylogenetic analyses place the new species in dictyostelid Group 3 (Rhizostelids) together with D. potamoides, with which it shares 65.8% identity for ITS and 96.6% for SSU. In this paper we describe a new species of cellular slime mold, Dictyostelium, ibericum, based on morphological and molecular characters. It is it small species with polar granules in its spores.
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34.
  • Romeralo, Maria, et al. (författare)
  • Evolution and Diversity of Dictyostelid Social Amoebae
  • 2012
  • Ingår i: Protist. - : Elsevier BV. - 1434-4610 .- 1618-0941. ; 163:3, s. 327-343
  • Forskningsöversikt (refereegranskat)abstract
    • Dictyostelid social amoebae are a large and ancient group of soil microbes with an unusual multicellular stage in their life cycle. Taxonomically, they belong to the eukaryotic supergroup Amoebozoa, the sister group to Opisthokonta (animals + fungi). Roughly half of the similar to 150 known dictyostelid species were discovered during the last five years and probably many more remain to be found. The traditional classification system of Dictyostelia was completely overturned by cladistic analyses and molecular phylogenies of the past six years. As a result, it now appears that, instead of three major divisions there are eight, none of which correspond to traditional higher-level taxa. In addition to the widely studied Dictyostelium discoideum, there are now efforts to develop model organisms and complete genome sequences for each major group. Thus Dictyostelia is becoming an excellent model for both practical, medically related research and for studying basic principles in cell-cell communication and developmental evolution. In this review we summarize the latest information about their life cycle, taxonomy, evolutionary history, genome projects and practical importance.
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35.
  • Romeralo, Maria, et al. (författare)
  • Two new species of dictyostelid cellular slime molds from Alaska
  • 2010
  • Ingår i: Mycologia. - : Informa UK Limited. - 0027-5514 .- 1557-2536. ; 102:3, s. 588-595
  • Tidskriftsartikel (refereegranskat)abstract
    • In sampling soils to survey dictyostelid cellular slime molds in Alaska we encountered two groups of isolates that have morphologies that differ from any previously described species within their group. We sequenced the nuclear small subunit ribosomal RNA gene (SSU rDNA) of selected isolates from the two groups and found sequences from both groups to be distinct from all previously described dictyostelid sequences. Phylogenetic analyses place one novel species in dictyostelid Group 2 and the other in Group 4 (Schaap et al. 2006). In this paper we formally describe as new these two species of cellular slime molds, Dictyostelium ammophilum sp. nov. and Dictyostelium borealesp. nov., based on the combination of morphological and molecular characters.
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36.
  • Sheikh, Sanea (författare)
  • Genomics of Sorocarpic Amoebae
  • 2017
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Sorocarpy is the aggregation of unicellular organisms to form multicellular fruiting bodies (sorocarps). This thesis is about the two best-known groups of sorocarpic amoebae, Dictyostelids and Acrasids.Paper I describes assembly and analysis of a multigene dataset to identify the root of the dictyostelid tree. Phylogenetic analyses of 213 genes (conserved in all sequenced dictyostelid genomes and an outgroup) place the root between Groups 1+2 and 3+4 (now: Cavenderiaceae + Acytosteliaceae and Raperosteliaceae + Dictyosteliaceae). Resolution of the dictyostelid root made it possible to proceed with a major taxonomic revision of the group.Paper II focuses on the taxonomic revision of Dictyostelia based on molecular phylogeny and SSU ribosomal RNA sequence signatures. The two major divisions were treated at the rank of order as Acytosteliales ord. nov. and Dictyosteliales. The two major clades within each of these orders were given the rank of family. Twelve genera were recognized. This is the first revision of a major protist taxon using molecular signatures and offers guidelines for taxonomic revision of protist groups where morphology is insufficient. Paper III presents the mitochondrial genome (mtDNA) of Acrasis kona. Over a quarter of the genome consists of novel open reading frames, while 16 genes present in the mtDNA of its relative, Naegleria gruberi, are missing. We identified many of these genes in the A. kona nuclear DNA, and used phylogenetic analyses to show that most of these genes arose by transfer from mtDNA. Paper IV presents the nuclear genome of A. kona, the second genome sequence of a free-living excavate. The 44 Mb genome has 15,868 open reading frames of which 4,987 are novel. A surprising number of genes are most similar to homologs in distant relatives, suggesting acquisition by horizontal gene transfer (HGT). Most HGT candidates are expressed and many constitute multi-gene families and/or have acquired introns and membrane targeting sequences. Strong HGT candidates include some genes essential to development and signaling in Dictyostelia. Flagellar motility and meiosis genes are also present and conserved, suggesting cryptic flagellar and sexual stages.
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37.
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38.
  • Sheikh, Sanea, et al. (författare)
  • Root of Dictyostelia based on 213 universal proteins
  • 2015
  • Ingår i: Molecular Phylogenetics and Evolution. - : Elsevier BV. - 1055-7903 .- 1095-9513. ; 92, s. 53-62
  • Tidskriftsartikel (refereegranskat)abstract
    • Dictyostelia are common soil microbes that can aggregate when starved to form multicellular fruiting bodies, a characteristic that has also led to their long history of study and widespread use as model systems. Ribosomal RNA phylogeny of Dictyostelia identified four major divisions (Groups 1–4), none of which correspond to traditional genera. Group 1 was also tentatively identified as sister lineage to the other three Groups, although not consistently or with strong support. We tested the dictyostelid root using universal protein-coding genes identified by exhaustive comparison of six completely sequenced dictyostelid genomes, which include representatives of all four major molecular Groups. A set of 213 genes are low-copy number in all genomes, present in at least one amoebozoan outgroup taxon (Acanthamoeba castellanii or Physarum polycephalum), and phylogenetically congruent. Phylogenetic analysis of a concatenation of the deduced protein sequences produces a single topology dividing Dictyostelia into two major divisions: Groups 1 + 2 and Groups 3 + 4. All clades in the tree are fully supported by maximum likelihood and Bayesian inference, and all alternative roots are unambiguously rejected by the approximately unbiased (AU) test. The 1 + 2, 3 + 4 root is also fully supported even after deleting clusters with strong individual support for this root, or concatenating all clusters with low support for alternative roots. The 213 putatively ancestral amoebozoan proteins encode a wide variety of functions including 21 KOG categories out of a total of 25. These comprehensive analyses and consistent results indicate that it is time for full taxonomic revision of Dictyostelia, which will also enable more effective exploitation.
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39.
  • Urbina, Hector, 1977-, et al. (författare)
  • Specificity in Arabidopsis thaliana recruitment of root fungal communities from soil and rhizosphere
  • 2018
  • Ingår i: Fungal Biology. - : Elsevier BV. - 1878-6146 .- 1878-6162. ; 122:4, s. 231-240
  • Tidskriftsartikel (refereegranskat)abstract
    • Biotic and abiotic conditions in soil pose major constraints on growth and reproductive success of plants. Fungi are important agents in plant soil interactions but the belowground mycobiota associated with plants remains poorly understood. We grew one genotype each from Sweden and Italy of the widely studied plant model Arabidopsis thaliana. Plants were grown under controlled conditions in organic topsoil local to the Swedish genotype, and harvested after ten weeks. Total DNA was extracted from three belowground compartments: endosphere (sonicated roots), rhizosphere and bulk soil, and fungal communities were characterized from each by amplification and sequencing of the fungal barcode region ITS2. Fungal species diversity was found to decrease from bulk soil to rhizosphere to endo-sphere. A significant effect of plant genotype on fungal community composition was detected only in the endosphere compartment. Despite A. thaliana being a non-mycorrhizal plant, it hosts a number of known mycorrhiza fungi in its endosphere compartment, which is also colonized by endophytic, pathogenic and saprotrophic fungi. Species in the Archaeorhizomycetes were most abundant in rhizosphere samples suggesting an adaptation to environments with high nutrient turnover for some of these species. We conclude that A. thaliana endosphere fungal communities represent a selected subset of fungi recruited from soil and that plant genotype has small but significant quantitative and qualitative effects on these communities.
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