Lecture 2: Describing Microbial Diversity: the ... - MCD Biology

Lecture 2: Describing Microbial Diversity: the Changing Paradigm
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General reading: S&F 638-648 (rudimentary phylogeny);
Pace, “Microbial Diversity and the Biosphere,” Science 276:734-740 (1997) (Website);
Pace, “Mapping the Tree of Life: Progress and Prospects,” Microbiol. Mol. Biol. Rev. 73:565-576 (2009)
(Website).
Baum, D.A., S.D. Smith and S.S. Donovan, “The Tree Thinking Challenge,” Science 310:979-980 (2005)
(with supporting info quiz, Website).
1. Traditional taxonomy (classification) of microbes is in a mess -- more on this later -- but at this stage we are
stuck with it for traditional reasons.
The traditional (Linnaean) classifications allocate organisms into “taxa” (s. taxon):
kingdom/phylum (division)/class/order/family/genus/species
However, these boundaries fall apart in the complexity of microbial diversity (and most phyla outside of animals;
“species” even fails in plants).
A. A “natural” taxonomy would be based on evolutionary relatedness:

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Thus, organisms in same “genus” (a collection of “species”) would have similar properties in a fundamental
sense; the representatives of a “species” are expected to share most properties with other representatives of
that species, perhaps with specialized gimmicks (“subspecies”, serovars, etc.).
1. A relatedness group at any level is a “clade”, a natural (phylogenetic) grouping. Representatives of a
clade are more closely related to one another than to any “outgroup” (non-clade) organism. “Phylotype” would
refer to the collection of organisms that make-up the clade, the phylogenetic type.
2. The question of what is a microbial “species” has recently become really complex with the revelation of
the concept of “pangenome” (text, p. 651-2). More later on this important topic, one issue in the description of
“microbial diversity”.
B. A natural taxonomy of macrobes has long been possible:
Large organisms have many easily distinguished features, e.g. morphology, body-plans and developmental
processes, that can be used to describe hierarchies of relatedness.
C. Microbes usually have few distinguishing properties that relate them, so a hierarchical taxonomy mainly was
not possible until molecular comparisons (not only sequence) came along.
2. Recent advances in molecular phylogeny have changed the picture a lot: we now have a relatively non-
subjective, semi-quantitative way to view “biodiversity”, in the context of phylogenetic “maps” - evolutionary trees.

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A. Slowly evolving molecules (e.g. rRNA) used for large-scale structure; more rapidly evolving (“fast-clock”)
molecules for fine-structure.
B. But always remember: an organism is much more than one gene; rRNA tells you about the evolution of
the genetic and basic cellular machinery, the equivalent of the “body plan” sought by the early
evolutionists.
3. A culmination is “The Big Tree” - a molecular coordinate system based on rRNA that relates all of life - below.
A. BUT, the literature, language (e.g. “species”) and formal nomenclature, of biology however, remain solidly
rooted in the tradition of Linnaeus at this time. (You have to call them something!)
B. First, an overview of phylogenetic perspective on microbial (indeed, biological) diversity, then a brief look at
other methods used to characterize microbes.
PHYLOGENETIC DIVERSITY
1. Phylogenetic relationships provide a “natural classification” of organisms (Darwin’s dream – “Our classifications
will come to be, as far as they can be so made, genealogies.”): Phylogenetic relationships are the only way to

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understand the evolutionary process. But, you need a metric – some property that provides a quantitative
measure of the extent of relatedness of different organisms.
A. Note that phylogenetic relationships can be “predictive”, not only “descriptive”: We can predict (some)
properties of organisms based on the properties of their relatives – the principle: representatives of a
particular phylogenetic group are expected to have the properties that are common to the group.
e.g. -- We know a lot about Escherichia coli, but relatively little about Chromatium vinosum:
1) E. coli is a chemoheterotroph, whereas C. vinosum is a photoheterotroph. (What do these terms
mean?) Classical physiological treatment would have considered these organisms wildly different. From
gene-sequence comparisons we know they are fairly close relatives (!-group of proteobacteria).
2) Thus, even though one eats glucose and the other light, we can predict that the biochemical underpinnings
are similar -- protein-synthesizing machinery (antibiotic-sensitivity), DNA replication machinery, amino acid-
synthesizing machinery, nucleotide biosynthesis, etc. Because of the close phylogenetic relationship, we
expect to be able to swap many genes between these two superficially disparate organisms.
3) The predictive value of phylogeny is essential for interpreting environmental sequence surveys.

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B. Phylogenetic perspective allows rational selection of model systems -- sometimes you need a model organism
to gain perspective on a more difficult system.
1) e.g. use of a model non-pathogen can provide safe study of a pathogen -- if it is a close relative. (What
means pathogen??)
e.g. Bacillus cereus and Bacillus anthracis: same organism except that the latter contains a few
pretty nasty genes for animals.
2) Or can provide a simple system for a complex one -- e.g. for plants, what to use as a model? Chlorella?
Chlamydomonas? Euglena?
Molecular phylogenetic studies say Chlorella or Chlamydomonas, both of which are in the plant relatedness
group (“clade”) -- Euglena is a trypanosome!
3) e.g. if you want to study the detail of an uncultivatable symbiont, identify a cultivatable free-living form. E.g.
the Riftia symbiont – how to choose the model?
C. Phylogenetic perspective even on large organisms is quite recent -- ~150 years -- and on microbes only ca. 30
years. Many microbiology texts don’t have it and many (even most) general biology texts get it wrong! (Although
it’s getting better.)

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A Bit on the Evolution of Evolutionary Thought
2. Prior to the late 19th century, the concept of evolution was on the “evolutionary ladder”:
Man
"
Apes
"
Marsupials
"
Reptiles
"
Amphibia
"
Fish
"
Invertebrates
"
Plants
"
Fungi
"
Leewenhoek’s “animacules”

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Indeed, the lexicon of biology still deals in “higher and lower” eucaryotes (I try not to use these terms -- they are
dumb), “missing links” (no such thing exists), and “primitive” organisms (no such thing today).
A. In its milieu, E. coli is as highly evolved as are we. E. coli is simple (~5#106 bp genome), we are complex
(~3#109 bps); complexity has nothing to do with “evolutionary advancement”.
B. Lineages evolve by diversification, "radiation", not “progression”. (!!)
C. There is no such thing as a “primitive” organism alive today. Simple, yes, but still a finely honed product of 4
billion years under the selective hammer of the niches that it and its progenitors have occupied.

3. By the late 1800s the concept of “evolutionary trees” was on the
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table -- e.g. Ernst Haeckel, 1866 (Note that Darwin’s “Origin of
Species” was first presented in 1858).
Note “kingdoms” of Plants, Animals, Protists (non-plants and
animals, mostly microbial), and “monera” (“procaryotes”) at the
base.
4. The conceptual basis for biological diversity was pretty much stalled
at the Haeckel stage for the next century -- and still is in many/most
general texts of biology. One current articulation is the “five
kingdoms of life”, here taken from the 1969 Science lead article that canonized 5-kingdoms; there are many
popular versions of the five-kingdoms notion, which dominates textbooks currently.

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A. Still, “monera” (mostly termed “procaryotes” by this time) at the origin and progression up a ladder of sorts. But
it still is the Haeckel tree in essence.
B. Note much other subjectivity -- e.g. why do “fungi” get to be a “kingdom”, and not e.g. Alveolates (inc. ciliates,
dinoflagellates) or Stramenopiles (inc. diatoms, brown algae, oomycetes)? [I guess because mushrooms are
large, sometimes?]
C. Note some subtleties by this time:

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1) Chloroplasts recognized as derived from "Blue-Green algae" (now “cyanobacteria” – one of ~100 bacterial
“phyla” (“divisions”, deepest clades in bacterial domain).
2) Mitochondria were thought probably derived from some sort of bacteria.
Note that the “endosymbiotic” origins for the organelles had been in the air since the 19th century -- for
comprehensive overview of this history (and controversy) see J. Sapp, “Evolution by Association: A History of
Symbiosis” (Oxford, 1994).
D. Still, there are many problems with this Five-kingdoms story:
1) Relationships among microbes, both “procaryote” and eucaryote were speculative, at best
2) There were no criteria to relate organisms between “kingdoms” (even between e.g. phyla of animals)-- a
universal phylogeny was impossible.
3) Implicit timeline remained -- “procaryotes”, “protists” “primitive.”

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4) Supposition that the eucaryotic nucleus was derived from a “procaryote” progenitor turned out to be
fundamentally incorrect (the often-cited date in textbooks, and even some of the current scientific literature, of
1.5 billion years ago for the origin of eucaryotes is utter B.S.) -
5) Studies in “molecular phylogeny” over the past two decades have changed the “paradigm” significantly. (ala
Thomas Kuhn -- “The Structure of Scientific Revolutions”)
E. By comparing macromolecular sequences, one can extract evolutionary relationships -- “evolutionary distances”
-- between organisms. The basic notion is that sequence change = evolutionary distance.
5. The goal of molecular phylogeny is to relate molecules (hence in principle organisms) quantitatively, so as to
reconstruct their evolutionary histories, e.g. as a “phylogenetic tree.”
A. There are many ways to "relate" molecules. Some subjective ways are:
e.g. immunologically (fractional gross reactivity)
e.g. DNA-DNA “heterologous hybridization” (more below)
But these are difficult (impossible) to quantify precisely in terms of relationships.

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B. The best way is by direct comparison of sequences of nucleic acids or proteins. This provides “precise”
numbers for defining relationships between molecules -- and, ideally, organisms.
6. For “orthologous” (of common ancestry and function) nucleic acid (or protein) sequences:
A. Consider:
--Organism X • • • AGCUGCCAGU • • •
X XX
--Organism Y • • • AACCCCCAGU • • •
"DNA OR RNA?
Sequence X is 70% identical to Sequence Y,
Fractional identity is 0.7
Fractional difference is 0.3 (1-0.7)
1) Note the terms: “homologous” = of common ancestry; “orthologous” = of common ancestry and function.
2) Note that the term “homology” is commonly used incorrectly when “identity” is meant. Note that
nucleotide sequences are not “##% similar”, they are “##% identical”; protein seqs, on the other hand, can
be “similar”. (How is that?)

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3) You cannot meaningfully compare sequences unless they are "homologous" -- of common ancestry.
Homologous sequences are not necessarily identical – indeed probably aren’t in different organisms;
identical sequences are not necessarily homologous, particularly if short (e.g. promoters, translation
punctuation, etc.).
B. Align sequences (big deal – more later) and do difference (1-identity) count for all pairs of organisms
considered. This difference count is a measure of the extent of evolution - evolutionary distance - separating the
pairs of organisms.
e.g. with organisms A,B,C,D and E:
C. To build relationships, construct a “difference matrix” for organisms A-E:

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A B C D E
A $ 0.1 0.2 0.2 0.4 Fractional Difference
B 0.9 $ 0.2 0.2 0.4
C 0.8 0.8 $ 0.1 0.4
D 0.8 0.8 0.9 $ 0.4
E 0.6 0.6 0.6 0.6 $
Fractional Identity
Can relate in a “tree”- like figure, a “dendrogram:”

Note that organism-to-node
“distance” is 1/2 of organism-
to-organism “distance
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Note that this (or any other) tree is a single dimension -- you can rotate around any node and have the same
topology in an evolutionary sense.
1) It is common to see sequence-divergence presented in terms of time, but this is not legitimate unless you
have a fossil record with which to calibrate the line segment-lengths. “Clock speeds” of organisms vary and the
evolutionary clock (rate of change) is not constant, contrary to common supposition
2) The root of the tree, the line representing the common ancestral line, may or may not be the “deepest”
branch point: Most properly the tree should be drawn:

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OR
3) E, the “outgroup”, "roots" A/B/C/D

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Vignette – Tree-reading –
Baum, D.A., S.D. Smith and S.S. Donovan, “The Tree Thinking Challenge,” Science 310:979-980 (2005) (with
supporting info quiz).
1. Remember – phylogenetic trees have only a single dimension – along line segments.
A few trees from Baum et al.:

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End Vignette
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7. What molecule(s) to use for molecular phylogeny of organisms for mapping the Tree of Life?

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A. Doesn’t really matter so long as:
1. “Homologous” gene occurs in all organisms considered
a. More specifically, you need to know that the genes are “orthologs,” not “paralogs”
b. “Orthologs” share ancestry and retain the same function in the different organisms
c. “Paralogs” result from an ancestral duplication, with potentially different functions taken-on subsequent
to the duplication – duplications produce “gene families”.
d. For instance, the % and & globins are a gene family; they have ancient common ancestry -- the % - type
and & - type globins have evolved independently since the ancestral duplication:
% - globins are orthologs; & - globins are orthologs.
% and & globins are paralogs
e. The tree of the gene family would look like:

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f. If you did not keep your orthologs and paralogs straight (sometimes a tough call) when you build the
dataset, you might get some most unexpected (and incorrect) trees, e.g.: You miss the true topology with the
restricted data set.
better.
Human %-globin
Mouse %-globin
Frog %-globin
Human &-globin
Mouse &-globin
Frog &-globin
Human (%-globin)
Frog (%-globin)
Mouse (&-globin)
2. You need a sufficient number of nucleotides (or amino acids) to be statistically significant -- more is always

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3. Changes must span the evolutionary distance inspected -- i.e., compared sequences must not be
randomized.
4. No lateral transfer -- the evolution of the gene must reflect the evolution of the cells considered.
a. Genes that are known to be derived from lateral transfer are called “xenologs.” If you are interested
in metabolic genes, there is a good chance, at least among bacteria, that you are dealing with
genes or pathways that potentially can move between different organisms.
b. e.g. penicillinase and other commonly transferred antibiotic resistance genes (scary!).
c. e.g. Rhizobium symbiotic (leguminous plants) N2-fixation.
5. Note the evident impact of lateral transfers throughout evolution. Much of microbial physiological diversity
(probably) is dependent on laterally transferred genes.
a. Note also that portions of genes can transfer (e.g. with “two- component” systems) so that
“homologous” blocks of sequence can show-up in functionally unrelated genes. Formation of
intralineage "gene families" also results in mixing-up functional modules of macromolecules.
b. How to detect xenologs?

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B. When “molecular phylogeny” first got started, considerable work was done with protein sequences, e.g.
cytochrome C, hemoglobin.
1. But proteins are hard to get and sequence; it is now easier to isolate/sequence genes.
2. Most protein genes are “shallow” clocks, the result of relatively recent evolution; e.g. E. coli doesn’t have
hemoglobin.
8. Choice of molecules for comprehensive (all organisms) phylogeny -- ribosomal RNAs (rRNAs).
A. Ribosome -- carries out protein synthesis
Small subunit Large subunit
S L “23S” rRNA (LSU): 3000-5000nt
“16S”-“18S” (SSU) rRNA: 1500-2000nt “5S” rRNA - 120 nt
ca. 25 proteins ca. 30-40 proteins
B. rRNAs present in all organisms and the major organelles (mitochondria and chloroplasts).

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C. Highly conserved throughout evolution; e.g., ca. 50% identity between E. coli and human SSU rRNAs over
alignable nt
1. Length variation between molecules may cause problems; must consider only “homologous” nt (the
alignment problem - more later).
2. Note that rRNA, while useful for distantly related organisms, is not good for establishing (resolving) close
relationships: it is too conservative. Consequently, close relatives have too few differences to be reliable.
e.g. human/apes/mice have the same rRNA seqs.
e.g. E. coli and Yersinia pestis (causes Black Plague) have essentially the same rRNA seqs.
D. The rRNA genes are large enough for reasonable statistics (more later on this).
E. First used for all-life phylogenetic trees by Carl Woese (University of Illinois).
For historical overview; Pace et al. “Phylogeny and beyond: scientific, historical and conceptual significance
of the first tree of life.” Proc. Natl. Acad. Sci. 109:1011-1018, 2012.
9. Given sequences of multiple organisms (>2 million now available, but spotty representation of diverse phyla –
MMBR reference):
$Align seqs., count number of differences -- is some measure of evolutionary change/distance

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$”Correct” for multiple and back mutations – the number of changes you count is always less than the number of
mutations that have occurred.
$Computer-fit pairwise “evolutionary distances” to best-fit overall tree topology.
(More detail on all this below)
10. The “Big Tree” emerges: Outlines first seen by Woese in 1977.
A. Note that Woese did not have tree-building methods now available. In fact, he did not have full sequences,
only short “signature” oligonucleotides (more on “signatures” later.

B. In essence, the tree is a quantitative map of evolutionary relatedness, a comprehensive map of biological
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diversity - - rRNA “sequence space”.

C. Indeed, the tree is a quantitative estimate – a metric - for that slippery concept, amount of evolution. The
“amount of biological diversity” (for any particular molecule) might be the summation of all unique line segment-
lengths in a comprehensive tree.
11.Some lessons from the Big Tree:
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A. There was a single origin for terrestrial type of life -- all life forms are related.
B. Three “primary lines of evolutionary descent” -- ”Domains” -- “ur-kingdoms”: Eucarya (eucaryotes),
Bacteria, Archaea (originally called “archaebacteria”, but the name was changed when it became clear that
the things aren’t bacteria.)
1. Sometimes see referred to as “kingdoms,” but usage in this context is probably not a good idea -- too
historically loaded.
2. You can inject "time" into tree, but sequence change is not necessarily linear with time - indeed, probably it
usually isn't.

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Bacteria
C. The eucaryote nuclear line of descent is as old as the archaeal line – eucaryotes have been around since
the beginning
Archaea
Eucarya
"Time"
2) The term “procaryote” is inappropriate in the light of the relationships – there isn’t any such group as
“prokaryote”. More on this issue later.
D. Are there still more domain-level divergences to be discovered??
E. Note that lines connecting organisms to nodes are not all the same length -- the evolutionary clock is not
constant between different lineages (e.g. Haloferax vs. Methanopyrus, Aquifex vs. Bacillus, Eucarya in general vs.
any representative of Archaea or Bacteria)

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1) The rate of evolution is not even necessarily the same for a particular lineage at all stages in the evolution
of the line, e.g. Agrobacterium vs. mitochondrion
2) Note domain-level tendencies:
Eucarya -- fast clocks
Archaea -- slow clocks
Bacteria -- intermediate rates of evolution
3) Because of variable rates, estimating time from sequence change is chancy--even fatuous--without some
sort of calibration, a correlation between the Tree and some datable geological event.
F. Note that the phylogenetic space occupied by multicellular eucaryotes is shallow and limited, but enormously
diverse in morphological (less biochemical) phenotype. Is this a consequence of large, highly plastic
genomes?
1) Note, however, that the “typical” eucaryote is microbial and has a small genome, e.g. Saccharomyces
cerevisiae at 13.5 # 106 bps. (vs, E. coli at ~4.2 # 106 bps; Calothrix [a cyanobacterium] at ~12.5 # 106 bps;
human at ~3.2 # 109 (mostly garbage?) bps; Methanococcus jannaschii at ~1.7 # 106 bps.)
G. The rRNA (and other molecular) data prove that mitochondria and chloroplasts were of bacterial origin (the
bacterial phyla [“divisions”] “proteobacteria” and “cyanobacteria,” respectively).

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H. Note how deeply divergent are Giardia, Trichomonas and Vairimorpha in the eucaryotic line. These
organisms lack mitochondria, so may have diverged from the main eucaryal line of descent before the mitos
came in.
1) It turns out they, like all eucaryotes have some bacterial (and archaeal) genes, but it is not clear where
or when they got them. Some folks think this resulted from mitochondria import but this is questionable.
12. The three-domain Big Tree is an “unrooted” tree -- you don’t know where is the ancestral node. You need
an “outgroup” to “root” the tree, and a universal tree has no outgroup.
A. However, the Tree can be rooted using “paralogs” that arose from duplication before the last common
ancestor:
e.g. translation factors EF-TU and EF-G
ATP synthase subunits % and &
tRNAs met-initiator and met-elongator
These paralogs occur in all three domains, so presumably arose before the last common ancestor. Each
yields the 3-domain tree upon analysis, so you can use tree with one paralog to root the tree with the other.
(You will do this in the Mol Phy Workshop.) All concur that the relationships are:

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Archaea
Eucarya
Bacteria
Archaea
Eucarya
Bacteria
This indicates that the root of the Big Tree is (presumably deep) on the bacterial line of descent.
B. This means also that Eucarya and Archaea shared common history after divergence from Bacteria
1) This explains many similarities between archaeal and eucaryal basal machineries.
e.g. similar transcription machineries; Archaea and Eucarya use TATA-binding proteins whereas Bacteria
use ' factors for specification of transcription initiation.
e.g. Archaeal and eucaryal DNA-synthetic machineries are far more like one another than either is to
bacteria (for overview, two recent review books are Garret and Klenk, “Archaea: Evolution, Physiology,
and Molecular Biology”, 2007. Cavicchioli, Archaea: Molecular and Cellular Biology, 2007).
13. Note that the Big Tree shown is a limited set of specific organisms: > 2 million SSU sequences are now
available.
A. Several databases of curated rRNA sequences e.g. RDP II, GreenGenes, SILVA, of course GenBank (raw
sequence - not aligned).

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You can download trees, carry out functions, get programs, etc.
14. A few domain-level trees for reference.
15. Note recent expansion of known bacterial diversity (also next page)
A. The lines indicate relatedness groups of bacteria, the phylogenetic “phyla” or “divisions” of bacteria (referred
to as “kingdoms” by Woese). There is no formal taxonomic status of these phyla at this time:

B. ca. 100 phyla identified so far by rRNA sequence. Only ca. 25 contain cultivated representatives (bold lines; non-
bold have no cultivated representatives). Only ~8-10 have significant cultured representation.
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1) Uncultivated organisms in the environment can be identified by obtaining rRNA genes without cultivation:
environmental sample ( isolate total DNA ( clone rRNA genes( sequence
(more later)
You know that the organism is there, and get some idea of abundance. How to get more information?
2) Most of the environmental sequences that are abundant in the environment are poorly represented by
cultivars, or not represented at all, e.g.:
The Acidobacterium group contains only a few cultivars, but is very abundant in many environments.
The“OP11” group is very abundant in anoxic environments at low and high temperatures, but has no
cultivated representatives
A tree with some names of the better-known groups is the following:

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C. Most of what we know about bacteria is based on studies of organisms representing only a few phyla:
$ Proteobacteria (E. coli, Pseudomonas spp., “purple photosynthetic bacteria”) - the classic "Gram
negative" group.

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$ Firmicutes (aka “Low G + C Gram Positive bacteria” [Bacillus, Clostridium,
Streptococcus, Staphylococcus, Lactobacillus)])
$ Actinobacteria (aka “High G + C Gram Positive bacteria” [Streptomyces,
Mycobacterium])
$ Cyanobacteria
D. Note the expansion in known bacterial diversity over the past few years!
16. Archaea:
A. Classically two groups (Crenarchaeota and Euryarchaeota) have cultivated representatives and recognition.
1) Crenarchaeota: Most cultivated types are high-temperature, but uncultivated low-temp. types are
abundant in the environment (detected by cloning rRNA and other genes – “metagenomics”)
a) Name “cren-” from the Greek for spring or fount, referring to the ostensible similarity of such organisms
to the earliest life (high temperature, using geothermal compounds for energy, e.g. H2 / S 0 -- more later)
2) Euryarchaeota: methanogens, extreme halophiles, many heterotrophs (more later):
a) Name from Greek “eury-” meaning “varied”, referring to variable
phenotypes, compared to cultivated crenarchaeota.

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B. Environmental sequences have swamped cultured sequences in the archaea, and the structure of the
archaeal tree currently is in a state of flux

17. Eucarya (Eucaryotes):
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A. Note that the microbial eucs are vastly more diverse than the popular three phyla (“kingdoms” --
fungi, plants and animal).
B. Note that the phylogeny of eucaryotes is a controversial (!!) place. rRNA always gives the same
pic as above. Protein gene trees are sometimes taken to represent various topologies and there is
little agreement in the field regarding the deepest branchings. For instance, a tree from Baldauf et al.
is the following:

18. The Large-scale Pic from the rRNA perspective is probably the least biased, but also is difficult to resolve at the
deepest branchings. What we see in the trees is biased by a lot to things, e.g. limited representation of known
diversity, treeing methods and nuances, “clockspeed” effects, others. See Pace 2009 for discussion.
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19. The three-domain tree is seen for all molecules in the central information-processing machinery (DNA, RNA,
protein synthesis), the “core” genes of genetic transfer.
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A. When you consider molecules outside these core functions, however, e.g. carbohydrate metabolism, amino
acid metabolism, etc., relationships can become weird. The susceptibility of genes to transfer may depend
on whether or not the gene product has to interact specifically with other cellular components; “stand-alone”
gene products can transfer more readily if they aren’t required to interact specifically with other cellular
components. Some genes seem to have moved around in the very deep past, e.g. aminoacyl-tRNA
synthases. In phylogenetic analysis of these genes, some give “canonical” three-domain trees, while others
show evidence of transfers.
B. E.g. the canonical pattern, as seen in the Leu RS:

C. Others are decidedly noncanonical, e.g. Ile RS:
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D. Even enzymes in the same biosynthetic pathway may have somewhat different evolutionary histories, e.g. in
His biosynthesis:

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Archaea (in bold), EUCARYA (IN CAPS)
1) Numbers at nodes are “bootstrap” valves, the % of trees in multiple solutions that give that particular node
(more later on “bootstrap analysis”).
E. These “incongruencies” with the Big Tree are generally considered to be the results of “lateral transfer” of
genes between the vertical lines of descent. For lots more discussion and the meaning in the larger context of
biology see:
Woese, Olsen, Soll. Aminoacyl-tRNA synthetases , the genetic code and the evolutionary process.
Microbiol. Molec. Biol. Rev. 64:202-236 (2000)
20. What happened in evolution? It looks as though the “core genome,” reflected in e.g. rRNA, had genetic
continuity throughout evolution. A lot of other things got scrambled – but not systematically.
A. “Endosymbiosis” has involved more than organelles!
B. Note that lateral transfer in general tends to be “phylogenetically local” and idiosyncratic. It can, however,
have powerful influence in evolution (e.g. cyanobacterial photosynthesis).
21. Some scientists argue that the occurrence of lateral transfers screws up large-scale trees: e.g. Ford Doolittle:

Gary Olsen edits the Doolittle tree to a more realistic view:
What lateral transfer really means phylogenetically is that if you want to track organismic lineages you need to be
wary of xenologs (and paralogs!). Remember: Any molecular tree is just that, a tree of molecules, not organisms.
22. Vignette: The prokaryote issue.
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