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MIB200A at UCDavis Module: Microbial Phylogeny; Class 1

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Slides for discussion of papers by Woese and Fox and Hugenholtz et al.

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MIB200A at UCDavis Module: Microbial Phylogeny; Class 1

  1. 1. Class 1: MIB200 Biology of Prokaryotes Class #1: Introduction UC Davis, Fall 2019 Instructor: Jonathan Eisen 1
  2. 2. Questions? • Experience level reading scientific papers? • Experience level writing scientific papers? • Experience level discussing scientific papers?
  3. 3. Raff J. How to Read and Understand a Scientific Article 1. Begin by reading the introduction, not the abstract. The abstract is that dense first paragraph at the very beginning of a paper. In fact, that's often the only part of a paper that many non-scientists read when they're trying to build a scientific argument. (This is a terrible practice. Don't do it.) I always read the abstract last, because it contains a succinct summary of the entire paper, and I'm concerned about inadvertently becoming biased by the authors' interpretation of the results. https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  4. 4. 2. Identify the big question.
 Not "What is this paper about?" but "What problem is this entire field trying to solve?" This helps you focus on why this research is being done. Look closely for evidence of agenda-motivated research. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  5. 5. 3. Summarize the background in five sentences or less. What work has been done before in this field to answer the big question? What are the limitations of that work? What, according to the authors, needs to be done next? You need to be able to succinctly explain why this research has been done in order to understand it. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  6. 6. 4. Identify the specific question(s).
 What exactly are the authors trying to answer with their research? There may be multiple questions, or just one. Write them down. If it's the kind of research that tests one or more null hypotheses, identify it/them. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  7. 7. 5. Identify the approach. What are the authors going to do to answer the specific question(s)? Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  8. 8. 6. Read the methods section. Draw a diagram for each experiment, showing exactly what the authors did. Include as much detail as you need to fully understand the work. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  9. 9. 7. Read the results section. Write one or more paragraphs to summarize the results for each experiment, each figure, and each table. Don't yet try to decide what the results mean; just write down what they are. You'll often find that results are summarized in the figures and tables. Pay careful attention to them! You may also need to go to supplementary online information files to find some of the results. Also pay attention to: • The words "significant" and "non-significant." These have precise statistical meanings. • Graphs. Do they have error bars on them? For certain types of studies, a lack of confidence intervals is a major red flag. • The sample size. Has the study been conducted on 10 people, or 10,000 people? For some research purposes a sample size of 10 is sufficient, but for most studies larger is better. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  10. 10. 8. Determine whether the results answer the specific question(s). 
 What do you think they mean? Don't move on until you have thought about this. It's OK to change your mind in light of the authors' interpretation -- in fact, you probably will if you're still a beginner at this kind of analysis -- but it's a really good habit to start forming your own interpretations before you read those of others. 
 Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  11. 11. 9. Read the conclusion/discussion/interpretation section. 
 What do the authors think the results mean? Do you agree with them? Can you come up with any alternative way of interpreting them? Do the authors identify any weaknesses in their own study? Do you see any that the authors missed? (Don't assume they're infallible!) What do they propose to do as a next step? Do you agree with that? 
 Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  12. 12. 10. Go back to the beginning and read the abstract. 
 Does it match what the authors said in the paper? Does it fit with your interpretation of the paper? Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  13. 13. 11. Find out what other researchers say about the paper. Who are the (acknowledged or self-proclaimed) experts in this particular field? Do they have criticisms of the study that you haven't thought of, or do they generally support it? Don't neglect to do this! Here's a place where I do recommend you use Google! But do it last, so you are better prepared to think critically about what other people say. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  14. 14. Two papers for today Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 5088-5090, November 1977 Evolution Phylogenetic structure of the prokaryotic domain: The primary kingdoms (archaebacteria/eubacteria/urkaryote/16S ribosomal RNA/molecular phylogeny) CARL R. WOESE AND GEORGE E. Fox* Department of Genetics and Development, University of Illinois, Urbana, Illinois 61801 Communicated by T. M. Sonneborn, August 18,1977 ABSTRACT A phylogenetic analysis based upon ribosomal RNA sequence characterization reveals that living systems represent one of three aboriginal lines of descent: (i) the eu- bacteria, comprising all typical bacteria; (ii) the archaebacteria, containing methanogenic bacteria; and (iii) the urkaryotes, now represented in the cytoplasmic component of eukaryotic cells. The biologist has customarily structured his world in terms of certain basic dichotomies. Classically, what was not plant was animal. The discovery that bacteria, which initially had been considered plants, resembled both plants and animals less than plants and animals resembled one another led to a reformula- tion of the issue in terms of a yet more basic dichotomy, that of eukaryote versus prokaryote. The striking differences between eukaryotic and prokaryotic cells have now been documented to construct phylogenetic classifications between do Prokaryotic kingdoms are not comparable toeukaryoti This should be recognized by,an appropriate terminolog highest phylogenetic unit in the prokaryotic domain we should be called an "urkingdom"-or perhaps "pr kingdom." This would recognize the qualitative dist between prokaryotic and eukaryotic kingdoms and emp that the former have primary evolutionary status. The passage from one domain to a higher one then be a central problem. Initially one would like to know wheth is a frequent or a rare (unique) evolutionary event. It i tionally assumed-without evidence-that the euka domain has arisen but once; all extant eukaryotes stem common ancestor, itself eukaryotic (2). A similar prejudic for the prokaryotic domain (2). [We elsewhere argue (15
  15. 15. Two papers for today Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 5088-5090, November 1977 Evolution Phylogenetic structure of the prokaryotic domain: The primary kingdoms (archaebacteria/eubacteria/urkaryote/16S ribosomal RNA/molecular phylogeny) CARL R. WOESE AND GEORGE E. Fox* Department of Genetics and Development, University of Illinois, Urbana, Illinois 61801 Communicated by T. M. Sonneborn, August 18,1977 ABSTRACT A phylogenetic analysis based upon ribosomal RNA sequence characterization reveals that living systems represent one of three aboriginal lines of descent: (i) the eu- bacteria, comprising all typical bacteria; (ii) the archaebacteria, containing methanogenic bacteria; and (iii) the urkaryotes, now represented in the cytoplasmic component of eukaryotic cells. The biologist has customarily structured his world in terms of certain basic dichotomies. Classically, what was not plant was animal. The discovery that bacteria, which initially had been considered plants, resembled both plants and animals less than plants and animals resembled one another led to a reformula- tion of the issue in terms of a yet more basic dichotomy, that of eukaryote versus prokaryote. The striking differences between eukaryotic and prokaryotic cells have now been documented to construct phylogenetic classifications between do Prokaryotic kingdoms are not comparable toeukaryoti This should be recognized by,an appropriate terminolog highest phylogenetic unit in the prokaryotic domain we should be called an "urkingdom"-or perhaps "pr kingdom." This would recognize the qualitative dist between prokaryotic and eukaryotic kingdoms and emp that the former have primary evolutionary status. The passage from one domain to a higher one then be a central problem. Initially one would like to know wheth is a frequent or a rare (unique) evolutionary event. It i tionally assumed-without evidence-that the euka domain has arisen but once; all extant eukaryotes stem common ancestor, itself eukaryotic (2). A similar prejudic for the prokaryotic domain (2). [We elsewhere argue (16
  16. 16. Woese and Fox Background
  17. 17. 1. Begin by reading the introduction, not the abstract 2. Identify the big question. 3. Summarize the background in five sentences or less. 4. Identify the specific question(s). 5. Identify the approach. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  18. 18. Woese and Fox Background
  19. 19. Woese and Fox Background
  20. 20. Woese and Fox Background
  21. 21. Woese and Fox Background
  22. 22. Woese and Fox Background
  23. 23. Woese and Fox Background
  24. 24. Woese and Fox Methods
  25. 25. 6. Read the methods section. Draw a diagram for each experiment, showing exactly what the authors did. Include as much detail as you need to fully understand the work. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  26. 26. Woese and Fox Methods
  27. 27. Woese and Fox Methods What is ribosomal RNA?
  28. 28. Central Dogma 29
  29. 29. The Ribosome 30
  30. 30. Ribosomal RNA structure 31
  31. 31. Woese and Fox Methods Why Use ribosomal RNA for this?
  32. 32. Methods Question: Why Use rRNA for this? • Universal • Highly conserved functionally • Evolves slowly • Easy to extract and sequence • Can compare sequences between species and used to infer relationships
  33. 33. Woese and Fox Results
  34. 34. 7. Read the results section. Write one or more paragraphs to summarize the results for each experiment, each figure, and each table. Don't yet try to decide what the results mean; just write down what they are. You'll often find that results are summarized in the figures and tables. Pay careful attention to them! You may also need to go to supplementary online information files to find some of the results. Also pay attention to: • The words "significant" and "non-significant." These have precise statistical meanings. • Graphs. Do they have error bars on them? For certain types of studies, a lack of confidence intervals is a major red flag. • The sample size. Has the study been conducted on 10 people, or 10,000 people? For some research purposes a sample size of 10 is sufficient, but for most studies larger is better. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  35. 35. 8. Determine whether the results answer the specific question(s). 
 What do you think they mean? Don't move on until you have thought about this. It's OK to change your mind in light of the authors' interpretation -- in fact, you probably will if you're still a beginner at this kind of analysis -- but it's a really good habit to start forming your own interpretations before you read those of others. 
 Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  36. 36. Woese and Fox Results
  37. 37. Woese and Fox Results: SAB Table For 13 Species
  38. 38. Woese and Fox Results: Kingdom #1
  39. 39. Type to enter text Woese and Fox Results: SAB Table For 13 Species
  40. 40. Woese and Fox Results: Kingdom #1
  41. 41. Woese and Fox Results: Kingdom 2
  42. 42. Type to enter text Woese and Fox Results: SAB Table For 13 Species
  43. 43. Woese and Fox Results: Kingdom 2
  44. 44. Woese and Fox Results: Kingdom 3
  45. 45. Type to enter text Woese and Fox Results: SAB Table For 13 Species
  46. 46. Woese and Fox Results: Kingdom 3
  47. 47. Woese and Fox Results: Three Kingdoms
  48. 48. Type to enter text Woese and Fox Results: SAB Table For 13 Species
  49. 49. Woese and Fox Results: Three Kingdoms
  50. 50. Type to enter text Woese and Fox Results: SAB Table For 13 Species Is There Another Way to View This?
  51. 51. Woese and Fox Discussion
  52. 52. 9. Read the conclusion/discussion/interpretation section. 
 What do the authors think the results mean? Do you agree with them? Can you come up with any alternative way of interpreting them? Do the authors identify any weaknesses in their own study? Do you see any that the authors missed? (Don't assume they're infallible!) What do they propose to do as a next step? Do you agree with that? 
 Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  53. 53. Woese and Fox Discussion
  54. 54. Woese and Fox Discussion
  55. 55. Woese and Fox Discussion
  56. 56. Woese and Fox Discussion
  57. 57. Woese and Fox Discussion
  58. 58. Woese and Fox Discussion
  59. 59. Woese and Fox Discussion
  60. 60. Class 1: MIB200 Biology of Prokaryotes Organisms without Nuclei Class #1: Introduction UC Davis, Fall 2019 Instructor: Jonathan Eisen 61
  61. 61. 62 Woese and Fox Abstract
  62. 62. 10. Go back to the beginning and read the abstract. 
 Does it match what the authors said in the paper? Does it fit with your interpretation of the paper? Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  63. 63. Woese and Fox Abstract
  64. 64. 11. Find out what other researchers say about the paper. Who are the (acknowledged or self-proclaimed) experts in this particular field? Do they have criticisms of the study that you haven't thought of, or do they generally support it? Don't neglect to do this! Here's a place where I do recommend you use Google! But do it last, so you are better prepared to think critically about what other people say. Raff J. How to Read and Understand a Scientific Article https://violentmetaphors.files.wordpress.com/2018/01/how-to-read-and-understand-a-scientific-article.pdf
  65. 65. 6
  66. 66. Two papers for today Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 5088-5090, November 1977 Evolution Phylogenetic structure of the prokaryotic domain: The primary kingdoms (archaebacteria/eubacteria/urkaryote/16S ribosomal RNA/molecular phylogeny) CARL R. WOESE AND GEORGE E. Fox* Department of Genetics and Development, University of Illinois, Urbana, Illinois 61801 Communicated by T. M. Sonneborn, August 18,1977 ABSTRACT A phylogenetic analysis based upon ribosomal to construct phylogenetic classifications between do 67
  67. 67. Hugenholtz et al
  68. 68. • Although we have yet to determine even the outlines of the bacterial tree, common threads are beginning to emerge that revise our current views of bacterial diversity and distribution in the environment. Hugenholtz et al
  69. 69. • These relatedness groups have variously been called “kingdoms,” “phyla,” and “divisions”; we use the latter term. • For the purposes of this review we define a bacterial division purely on phylogenetic grounds as a lineage consisting of two or more 16S rRNA sequences that are reproducibly monophyletic and unaffiliated with all other division-level relatedness groups that constitute the bacterial domain • We judge reproducibility by the use of multiple tree-building algorithms, bootstrap analysis, and varying the composition and size of data sets used for phylogenetic analyses. Hugenholtz et al
  70. 70. • Division-level nomenclature has not even been consistent between studies, so some divi- sions are identified by more than one name. For instance, green sulfur bacteria is synonymous with Chlorobiaceae; high- G C gram-positive bacteria is synonymous with Actinobacteria and Actinomycetales. Indeed, it probably is premature to standardize taxonomic rankings for bacterial divisions at this point when our picture of microbial diversity is likely still incomplete and the topology of the bacterial tree is still unresolved.
  71. 71. • Figure 1 represents the division-level diversity of the bacterial domain as inferred from representatives of the approximately 8,000 bacterial 16S rRNA gene sequences currently available. Although 36 divisions are shown in Fig. 1, several other division-level lineages are indicated by single environmental sequences (9, 21, 37), suggesting that the number of bacterial divisions may be well over 40.
  72. 72. FIG. 1. Evolutionary distance tree of the bacterial domain showing currently recognized divisions and putative (candidate) divisions. The tree was constructed using the ARB software package (with the Lane mask and Olsen rate-corrected neighbor-joining options) and a sequence database modified from the March 1997 ARB database release (43). Division-level groupings of two or more sequences are depicted as wedges. The depth of the wedge reflects the branching depth of the representatives selected for a particular division. Divisions which have cultivated representatives are shown in black; divisions represented only by environmental sequences are shown in outline. The scale bar indicates 0.1 change per nucleotide. The aligned, unmasked data sets used for this figure and Fig. 3 through 6are available from http:// crab2.berkeley.edu/pacelab/ 176.htm. 74
  73. 73. • Indeed, 13 of the 36 divisions shown in Fig. 1 are characterized only by environmental sequences (shown outlined) and so are termed “candidate divisions” new bacterial divisions • One of these candidate divisions, OP11, is now sufficiently well represented by environmental sequences to conclude that it constitutes a major bacterial group (see below). • Phylogenetic studies so far have not re- solved branching orders of the divisions; bacterial diversity is seen as a fan-like radiation of division-level groups (Fig. 1). The exception to this, however, is the Aquificales division, which branches most deeply in the bacterial tree in most analyses. •
  74. 74. 76
  75. 75. 77
  76. 76. 78
  77. 77. FIG. 2. Relative representation in selected cosmopolitan bacterial divisions of 16S rRNA sequences from cultivated and uncultivated organisms. Results were compiled from 5,224 and 2,918 sequences from cultivated and uncultivated organisms, respectively. 79
  78. 78. • The database of environmental rRNA sequences is compromised in resolving some phylogenetic issues by a large number of relatively short sequences. More than half of the sequences collated in Table 1 are less than 500 nucleotides (nt) long, which represents only one-third of the total length of 16S rRNA. This is due to an unfortunate trend in many environmental studies of sequencing only a portion of the gene in the belief that a few hundred bases of sequence data is sufficient for phylogenetic purposes. Indeed, 500 nt is sufficient for placement if some longer sequence is closely related ( 90% identity in homologous nucleotides) to the query sequence. In the case of novel sequences, 85% identical to known sequences, however, 500 nt is usually insufficient comparative information to place the sequence accurately in a phylogenetic tree and can even be misleading
  79. 79. Acidobacterium FIG. 3. Phylogenetic dendrogram of the Acidobacterium division. Names of cultivated organisms are shown in bold. The habitat source of each environmental sequence is indicated before the clone name. GenBank accession numbers are listed parenthetically. Subdivisions (see the text) are indicated by brackets at the right of the tree. Construction of the tree was as described for Fig. 1. The robustness of the topology presented was estimated by bootstrap resampling of independent distance, parsimony, and rate- corrected maximum-likelihood analyses as previously described (2). Distance and parsimony analyses were conducted using test version 4.0d61 of PAUP*, written by David L. Swofford. Branch points supported (bootstrap values of >75%) by most or all phylogenetic analyses are indicated by filled circles; open circles indicate branch points marginally supported (bootstrap values of 50 to 74%) by most or all analyses. Branch points without circles are not resolved (bootstrap values of <50%) as specific groups in different analyses. The scale bar indicates 0.1 change per nucleotide. 81
  80. 80. Verrucomicrobia FIG. 4. Phylogenetic dendrogram of the Verrucomicrobia division. Names of cultivated organisms are shown in bold. The habitat source of each environmental sequence is indicated before the clone name. GenBank accession numbers are listed parenthetically. Subdivisions (see the text) are indicated by brackets at the right of the tree. Tree construction and support for branch points was as described for Fig. 1 and 3, respectively. The scale bar indicates 0.1 change per nucleotide. 82
  81. 81. Green non sulfur FIG. 5. Phylogenetic dendrogram of the GNS division. Names of cultivated organisms are shown in bold. The habitat source of each environmental sequence is indicated before the clone name. GenBank accession numbers are listed parenthetically. Subdivisions (see the text) are indicated by brackets at the right of the tree. Tree construction and support for branch points was as d e s c r i b e d f o r F i g . 1 a n d 3 , respectively. The scale bar indicates 0.1 change per nucleotide. 83
  82. 82. OP11 FIG. 6. Phylogenetic dendrogram of the OP11 division. The habitat source of each environmental sequence is indicated before the clone name. GenBank accession n u m b e r s a r e l i s t e d parenthetically. Subdivisions (see the text) are indicated by brackets at the right of the tree. Tree construction and support for branch points was as described for Fig. 1 and 3, respectively. The four MIM clones and F78 clone are unreleased sequences generously made available to us by Pascale Durand (10) and Floyd Dewhirst (8). The scale bar indicates 0.1 change per nucleotide. 84
  83. 83. Conclusions • novelties are known as well, for instance, endospore formation by the low-G C gram-positive bacteria or axial filaments (endoflagella) in the spirochetes. Some biochemical properties evidently have transferred laterally among the divisions. For example, the two types of photosynthetic complexes, photosystem I (PSI) and PSII, are each distributed sporadically among the divisions, consistent with lateral transfer (3). Lateral transfer may also have resulted in combinatorial novelty among the divisions; PSI and PSII, for instance, apparently came together in the cyanobacteria to create oxygenic photosynthesis, with profound consequences to the biosphere (3). • Many more such division-specific qualities and cooperations should become evident at the molecular level as comparative genomics gives us a sharper phylogenetic picture of bacterial diversity.
  84. 84. • PCR and microbial community surveys possible issues • Where could this go “wrong”? •

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