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An Introduction to The Big Tree

So, what do you get if you use the ssu-rRNA molecular phylogenetic methods we've talked about with representatives of all kinds of organisms? You get this unrooted dendrogram:

Some features of this 'universal' molecular phylogenetic tree (The Big Tree):

Remember that all previous trees were subjective & qualitative. This tree is quantitative and objective, based on statistical analysis of gene sequences (in this case small subunit ribosomal RNA, but other appropriate molecules yield essentially the same result).

To everyones continuing surprise, there are 3 major, distinct evolutionary groups:

• Bacteria = eubacteria
• Archaea = archaebacteria
• Eukarya = eukaryotic nuclear/cytoplasmic lineage

Prokaryotes fill 2/3rds of the tree (2 of the 3 Domains). There are 2 separate groups of prokaryotes. Multicellular eukaryotes are a very small portion of evolutionary diversity - just the tip of one branch of the eukaryotes, not 3/5ths of evolutionary diversity as the 5-Kingdom scheme implies.

One of the most exciting outcomes of this method early on was the discovery of a new type of organism - the Archaea, These species had previously been scattered haphazardly amongst the bacteria. Phenotypically, these organisms are generally similar to Bacteria, but biochemically they are generally similar to Eukarya. They have changed less since their common ancestry than the other groups, and so more closely resemble our common ancestry - they are a kind of a 'missing link' between eukaryotes & Bacteria, and 'living fossils' of early life. Bacteria, however, are every bit as highly evolved (at least in terms of rRNA sequence divergence) as are eukaryotes.

The Bacteria turn out to be composed of a handfull of major groups and an ever-growing list of minor groups, all with Gram-negative type cell envelopes except one - the Gram-positive Bacteria are just one of the major branches. Most of the other Bacteria we're familiar with are members of the Purple Bacteria & relatives, also just a single one of the major branches.

Eukaryotes are as ancient a group as are the prokaryotic Domains, and eukaryotes did not evolve from either of the other groups. Interestingly, all of the known branches of eukaryotes are 'late' (there is a long bare branch connecting eukaryotes to the other Domains). There are no known primative eukaryotes or early branches in the eukaryotic group. It isn't clear how many main eukaryotic groups there might be - there are lots of described protists that have not been analyzed genetically. In fact, the eukaryotes are the least well-studied of the Domains in terms of molecular phylogeny.

The tree also offers final proof of the endosymbiont theory for the origin of mitochondria and chloroplasts. The mitochondria turn out to be purple Bacteria, and the chloroplasts are cyanoabacteria.

Bacteria

The main branches of Bacteria mostly arose at once in a "main radiation". There are just a few major groups ('Kingdoms') of Bacteria, related by this tree:

Most of the branches of Bacteria, and certainly most of the branches with lots of known and abundant species, radiation from a single point in the tree, the "main radiation". There are some earlier branches; these are minor groups with few species that are not generally abundant, and they are primative organisms (short branch length) and mostly thermophilic.

• Early, primative, thermophilic branches:
  • Aquifex/Hydrogenobacteria (including Thermocrinus, the pink filamentous mat former in Yellowstone hot springs)
  • Thermotoga
  • Green non-sulfur Bacteria (including Chloroflexus, the golden mat former in Yellowston hot springs)
• Main radiation:
  • Deinococci (including Thermus)
  • spirochaetes (common in the environment and as animal pathogens)
  • green sulfur Bacteria (green anaerobic sulfur-oxiding photosynthesizers - not very familiar to most)
  • Cytophaga/Bacteroids/Flavobacteria (Bacteroides is one of the abundant gut organism in humans - the aerobes are common but unfamiliar, they include the chitin/agar degraders we're isolating and the Amp-resistant Bacteria some of you have)
  • Planctomycetes (common but few are cultiavted & so are little understood)
  • Chlamydia (hopefully you know this one, but not too well - these are all a small, closely-related cluster of species)
  • Gram positive Bacteria (OK, now we're reached the hugely common groups - these are predominant in soil, and lots of pathogens/symbionts)
  • Cyanobacteria (these are the blue-greens, they carry out oxygenic photosynthesis and power the biosphere today)
  • Purple Bacteria (most Gram-negative Bacteria you'd think of are members of this group)
  And, of course, lots of other less well-known branches.

Archaea

The Archaea fall into two main phylogenetic groups, the Euryarchaea and the Crenarchaea. The Korarchaea are another group, but all we know about them is that they're there, from ssu-rRNA sequences fished out of the environment. None are in culture.

In this tree of the Archaea, the sulfur-metabilzing thermophiles are shown in orange, methanogens in green, and halophiles in red. The Euryarchaea are on top (Haloferax-Pyrococcus) and the Crenarchaea on bottom (Thermoproteus-Sulfolobus).

The Crenarchaea in culture are all thermophilic and are at least facultative sulfur-metabolizers. Some grow by sulfur oxidation, some by sulfur reduction, some by sulfur respiration, and some can switch from one to another depending on environmental conditions. Many of these organisms grow at low pH, and so they are also sometimes called the "thermoacidophiles". There are also lots of mesophilic crenarchaea, but none have been cultivated.

The Euryarchaea in culture fall into 3 broad phenotypic classes: sulfur-metabolizing thermophiles, methanogens, and extreme halophiles. The sulfur-metabolzing thermophiles amongst the euryarchaea braanch deeply in the tree and phenotypically resemble the crenarchaea; this means that this was probably the primitive phenotype of the Archaea. Methanogens make methane (CH4), usually from CO2 and H2, but some can use other 1-carbon compounds (CO, HCOOH, H3COH, etc) and one group can use acetate or methylamines without H2. Methanogens are extreme anaerobes, most not only require the absence of oxygen, but also extremely reduced environments. Methogens are the source of flammable swamp gas, intestinal gas, and a large fraction of natural gas. The extreme halophiles are the organisms that live in salt and soda lakes like the Dead Sea, Great Salt Lake, etc.

Eukarya

This branch represents the nuclear/cytoplasmic part of eukaryotes. Mitochondria are, as we know, descended from the purple Bacteria, and plastids from cyanobacteria. Notice the long branch connecting this branch to the Bacteria/Archaea - there are no known "early branches" of the Eukarya (unless you consider the Archaea to be just that). Also notice that, unlike the Bacteria, the early branches of the Eukarya are not primitive, but actually more highly derived than are the big, lumbering Eukarya. In fact, the Diplomonads (e.g. Giardia), Microsporidia, and Parabasalians represent a wide diversity of parasitic anaerobic eukaryotes that lack mitochondria. Whether they never had them (i.e. they branched off before the ancestor of the other eukarya acquired the mito) or had them and lost them is a matter of some debate.

Notice that nearly all of these branches are protists (unicellular eukaryotes)- the plants, animals and fungi represent only a small portion of this tree. In fact, it's even worse than this - even most of these creatures are microscopic, when you think about it. Of course you know this for fungi, but did you know that the vast majority of plants are unicellular - we call them the green algae. Likewise animals; the world is crawling with too-small-to-be-seen animals - rotifers, tardigrades, nemotodes (this is a huge and incredibly numerous group), microscopic crustaceans and other arthropods.

Rooting the Universal tree

Where in the 'Big Tree' is the common ancestor? To answer this, you would need to root the tree, but to root a tree you need an outgroup, and how do you get an outgroup for a universal tree?

The solution to this quandry lies in the fact that the trees generated in a molecular phylogenetic analysis are trees of gene sequences, not organisms. This distinction can be used to create a rooted phylogenetic tree of all organisms by using sequences that span a broader evolutionary breadth than do all known organisms.

This is done by using gene sequences from a gene family that split before the last common ancestor, e.g. ATPase subunits alpha vs beta, initiator vs elongator tRNA(Met), EF-Tu vs EF-G, any pair of related aminoacyl-tRNA-synthetases (e.g. Leu vs Ile).

When you do this, you end up with a dendrogram with two main clusters, one for each sub-type of sequence. The sub-tree from one member of the gene family serves as root for the other & vice versa !

The root turns out to be very close to the base of all three Domains, on the branch dividing the Bacteria from the Archaea & Eukarya. In other words, the Archaea and Eukarya are related, not the Bacteria & Archaea (i.e. prokaryotes), as most thought. This is being substantiated more & more by gene & especially genome sequences.

The Archaea are, therefore, an early, primative branch of the eukaryotic lineage. This is the reason for a lot of the interest in Archaea these days; these organisms probably resemble the ancestor of eukaryotes, and certainly have much to tell us about where eukaryotes (i.e. us) came from.

The Caveat

The fly in the ointment of molecular phylogenetic analysis, especially in attempts to sort out the deepest parts of the tree like this, is the issue of horizontal transfer. The trees we've been talking about as based in ssu-rRNA, and these are substantiated by a number of other highly conserved genes. However, we know that genes have "moved" from one organism to another across phylogeny - in fact, across the farthest reaches of the tree. We've been assuming that this gene flow is inconsequential, but is it? At the other end of the spectrum, if an organism's are mixtures of genes from a wide range of sources, do the even haave a specific geneology?

The current consensus (well, not really a consensus, it's a topic of hot debate) is that gene flow does exist, and is an important source of new information in microbial populations. However, the information-processing "core" of the cell is relatively resistant to this gene flow, and so this is what we're representing in these trees. However, there is reason to believe that horizontal transfer was more common in the past than it is today. In fact, the emergence of the Bacteria, Archaea, and Eukarya may actually represent the emergence of more-or-less independently evolving lineages from a prior amalgamated gene pool:

(from Ford Dolittle)

Molecular phylogenetic analysis of uncultivated organisms and populations

Most of what we know about the microbial world is taken from the properties of cultivated organisms. But most bacterial species are not readily cultivatable (as we will see in lab), so you inevitably end up examining unrepresentative organisms if you rely on cultivation - all you can study are the "weeds". Inferences from these species are unlikely to be realistic.

Molecular phylogenetic analysis can solve this problem, because cultivation is not required. Molecular phylogenetic analysis can be performed on isolated (cultivated or not) material, or even of microbial populations. Molecular phylogenetic analysis of uncultivated organisms is similar to analysis of cultivated species, but starts with DNA extracted directly from a purified sample or the environment, rather than from a pure culture:

1. Extract DNA directly from purified sample or an environmental sample
2. Amplify rRNA genes by PCR using universal primers
3. Clone, sequence, & tree the amplified genes
4. Use these sequences to design specific hybridization probes to go test the occurence, abundance, morphology, etc, of the specie in the population

Some uses of molecular phylogenetic analysis in microbial ecology:

• Identify the predominant microbial groups in a population
• Count sequences from various phylogenetic group to assess their abundance in the ecosystem
• Assess enrichments aimed at cultivating organisms previously identified by rRNA sequence.
• Use fluorescently-labeled rRNA sequences (oligonucleotides) as probes for the identification or enumeration of specific organisms or groups in environmental samples.
• Identification of unculturable organisms. e.g. the identification of uncultivatable pathogens in tissue samples.

Questions for thought

• In the big tree, most of the deep (early branching) species of eukaryotes are parasites - Giardia, microsporidia, trypanosomes, etc. Can you think of a reason why this might be?
• Do microbes actually have meaningful geneologies?
• Do molecular phylogenies represent how primative or 'advanced' an organism (not just the gene studied) is?
• Given than Bacteria and Archaea (and many eukarya, for that matter) do not reproduce sexually, how would you define species in these organisms?
• How could the properties of the three evolutionary groups be useful in predicting specific properties of the last common ancestor?
• It appears that less than 1% of living things have been characterized, and in only a very small fraction of these have ssu-rRNA sequences been determined. How might the Big Tree change as more and more ssu-rRNA sequence become available for analysis?
• Do molecular phylogenetic trees really represent biological 'diversity'? How would you define diversity?