Microbial Phylogenomics (EVE161) at UC Davis Spring 2016. Co-taught by Jonathan Eisen and Holly Ganz. Class 7: Era II: rRNA PCR and Major Group

1. Lecture 3:
EVE 161:
Microbial Phylogenomics
Class #7:
Era II: rRNA PCR and major groups
UC Davis, Winter 2016
Instructors: Jonathan Eisen & Holly Ganz

2. Where we are going and where we have been
• Previous lecture:
!6. Era II: rRNA from environment
• Current Lecture:
!7: Era II: PCR and major groups
• Next Lecture:
!8: Era II: rRNA ecology
!2

3. !3

5. • 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.

7. • 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.

8. • 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.

9. • 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.

10. 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.
!10

11. • 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.

12. !12

13. !13

14. !14

15. 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.
!15

16. • 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

17. Acidobacterium
FIG. 3.
Phylogenetic dendrogram of the Acidobacteriumdivision. 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.
!17

18. Verrucomicrobia
FIG. 4.
Phylogenetic dendrogram of theVerrucomicrobia 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.
!18

19. 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 described for Fig. 1and 3, respectively. The scale
bar indicates 0.1 change per nucleotide.
!19

20. OP11
FIG. 6.
Phylogenetic dendrogram of the OP11
division. 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 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.
!20

21. 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 ev- ident at the molecular level as comparative
genomics gives us a sharper phylogenetic picture of bacterial
diversity.

22. !22

23. Summary: The intent of this article is to provide a critical assessment of our current
understanding of life's phylogenetic diversity. Phylogenetic comparison of gene
sequences is a natural way to identify microorganisms and can also be used to infer
the course of evolution. Three decades of molecular phylogenetic studies with
various molecular markers have provided the outlines of a universal tree of life
(ToL), the three-domain pattern of archaea, bacteria, and eucarya. The sequence-
based perspective on microbial identification additionally opened the way to the
identification of environmental microbes without the requirement for culture,
particularly through analysis of rRNA gene sequences. Environmental rRNA
sequences, which now far outnumber those from cultivars, expand our knowledge
of the extent of microbial diversity and contribute increasingly heavily to the
emerging ToL. Although the three-domain structure of the ToL is established, the
deep phylogenetic structure of each of the domains remains murky and sometimes
controversial. Obstacles to accurate inference of deep phylogenetic relationships
are both systematic, in molecular phylogenetic calculations, and practical, due to a
paucity of sequence representation for many groups of organisms.
!23

24. Sequence uncertainty with depth in a phylogenetic tree.
Pace N R Microbiol. Mol. Biol. Rev. 2009;73:565-576
Sequence uncertainty with
depth in a phylogenetic tree.
Dashed line, not corrected for
unseen changes; solid line,
corrected for unseen changes
using the following estimation:
inferred sequence change
(Knuc) = −3/4 ln[1 − (4/3)D],
where D is the number of
changes counted (31).
!24

25. Chronological accumulation of SSU rRNA sequences.
Pace N R Microbiol. Mol. Biol. Rev. 2009;73:565-576
Chronological accumulation of
SSU rRNA sequences. The
data are derived from the
SILVA 98 SSU Parc database
(52) using the EMBL
taxonomic designations for the
sequences (66). The SILVA
SSU Parc database contains
rRNA sequences that are 300
or more nucleotides in length
and validated as rRNA with
RNAmmer (43). (A)
Accumulation of total,
archaeal, bacterial, and
eucaryal SSU sequences. (B)
Accumulation of rRNA
sequences from cultured and
environmental bacteria. (C)
Accumulation of rRNA
sequences from cultured and
environmental archaea.
!25

26. Sequences vs. Time
!26

27. Bacteria vs. Time
!27

28. Archaea vs Time
!28

29. A molecular ToL based on rRNA sequence comparisons.
Pace N R Microbiol. Mol. Biol. Rev. 2009;73:565-576
A molecular ToL based on
rRNA sequence
comparisons. The
diagram compiles the
results of many rRNA
sequence comparisons.
Only a few of the known
lines of descent are
shown.
!29

30. Named Phyla

31. Distribution of SSU rRNA among the top 12 bacterial phyla.
Pace N R Microbiol. Mol. Biol. Rev. 2009;73:565-576
Distribution of SSU rRNA
sequences among the top
12 bacterial phyla. Shown is
the SSU rRNA sequence
distribution in the SILVA 98
SSU Parc database (52)
among the bacterial phyla
(Ribosomal Database
Project taxonomy) (10)
containing the most rRNA
sequences.
!31

32. Archaeal rRNA trees with sequences available in 1993 and 2008.
Pace N R Microbiol. Mol. Biol. Rev. 2009;73:565-576
Archaeal rRNA trees with sequences available in
1993 and 2008. Archaeal SSU rRNA sequences
available in 1993 (classic archaeal tree) (A) and in
2008 (B) were used in maximum likelihood
bootstrap analysis with RAxML (64) as described
previously (56, 57). The boxes represent radiations
within the groups, with the long and short
dimensions reflecting the line segment lengths
within the groups. The sizes of the boxes reflect
sequence representation for the groups. The
numbers at the base of the boxes are bootstrap
percentages. The box labeled Environmental
“Euryarchaeota” is not a phylogenetically coherent
group.
!32

33. Archaeal rRNA trees with sequences available in 1993 and 2008.
Pace N R Microbiol. Mol. Biol. Rev. 2009;73:565-576
!33

34. Archaeal rRNA trees with sequences available in 1993 and 2008.
Pace N R Microbiol. Mol. Biol. Rev. 2009;73:565-576
!34

35. Distribution of SSU rRNA among the top 12 eucaryal phyla.
Pace N R Microbiol. Mol. Biol. Rev. 2009;73:565-576
Distribution of SSU rRNA
sequences among the top
12 eucaryal phyla. Shown
is SSU rRNA sequence
distribution in the SILVA 98
SSU Parc database (52)
among the eucaryotic
phyla (EMBL taxonomy
[66]) containing the most
rRNA sequences.
!35

36. • The articulation of an accurate universal ToL, a map of
life’s evolutionary course, is a lofty goal. Enormous
strides in the direction of that goal have been taken as the
molecular view of life has developed. The outlines of a
universal tree are in place; microbial classification can
aspire to a solid foundation based on sequence
comparisons; environmental sequences reveal a rich
world of unanticipated microbial diversity with significance
for the working of ecosystems.

37. • For all the progress, however, these successes also reveal how little
we really know about microbial diversity and, con- sequently, how
uncertain is our perception of life’s phylogenetic history at the
deepest levels. It is only clear at this time that we have merely
scratched the surface of an enormous microbial diversity in all the
domains, archaeal, bacterial, and eucaryal. To begin to understand
the scope of this diversity, continued phylogenetic survey of natural
ecosystems has a critical place among the large agendas of the
biological sciences. The results will continue to clarify and confound
and bring new insights to our understanding of the global biosphere.
Continued discovery of major microbial groups, new arenas for
research and resources, seems certain. It also seems certain that
future sequence acquisitions will continue to sharpen the molecular
view of the deepest branches in the ToL.
• The future of microbiology is bright.

38. Side issues
• Orthologs and Paralogs
• Unseen changes
• Testing trees
• What we do not know
!38

39. Rooting TOL Review

40. Microbiological Reviews 51:221

41. Simplified, Rooted Tree of Life