Update on PNAS flagellum paper

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Those who have been following the comments section of the first post on the PNAS flagellum paper, entitled “Stepwise formation of the bacterial flagellar system,” will see that there have been several developments: ScienceNOW at Science magazine has uncritically reported the PNAS paper’s all-flagellar-genes-came-from-one conclusion; Behe and other IDers are getting into the act, although they are so clueless they don’t really even understand why the PNAS paper is problematic; and PZ Myers and I have dropped hints that several of us PT bloggers are reaching the conclusion that this paper is looking worse, not better, after close examination. We will have more on the technical methodology issues in the next few days. For the moment I would just like to offer a simple response to some comments, and a simple but powerful reason that the “all core flagellum genes are descended from one ancestral gene” does not work.

First, the comments. Some commentators have reacted along the following lines: (1) maybe the paper isn’t so bad, just speculative; and/or (2) maybe I’ve misread the paper or its conclusion was poorly worded, and maybe the authors just meant to argue that some of the 24 core flagellar proteins were related, not all 24 proteins.

Unfortunately – and I mean unfortunately because I wish one of these options was true – neither idea is a supportable interpretation of the authors’ views. Have another look at Figure 3 from the Liu & Ochman paper:

  • The boxes contain the names of all 24 core flagellar proteins that the authors identified.
  • Each line represents a significant homology hit using pairwise BLAST. The claimed e-value cutoff for significance is 10-4 (e=0.0001), which is pretty conservative – researchers sometimes assign homology pretty confidently on e-values up to 10-2 (e=0.01).
  • The numbers represent the number of times, in 41 tested bacterial genomes, that the homology was found. For example, the already well-known homology between FliC (flagellin) and FlgL (flagellin/hook adapter) was recovered 41/41 times.
  • The blue lines represent homologies retrieved in E. coli K12, the grey lines hits retrieve in any of the 41 genomes.

In some ways, Figure 3 confirms conventional wisdom. The axial filament of the flagellum – the tube that extends from the basal body out to the filament – is made up of a series of proteins, approximately in this arrangement, with these copy numbers:

~9 copies of FliE (FliF-rod linker) ~6 copies of FlgB (inner rod) ~6 copies of FlgC (middle rod) ~6 copies of FlgF (middle rod) ~26 copies of FlgG (outer rod) ~130 copies of FlgE (hook – the curvy part in most flagellum diagrams) ~11 copies of FlgK (first hook-filament linker) ~11 copies of FlgL (second hook-filament linker) ~20,000 copies of FliC (flagellin, makes up the flagellar filament)

The long tube that these proteins form is called the “axial filament” and the proteins as a group are “axial proteins.”

Now, if you remember Table 1 from Pallen and Matzke 2006, a simple protein BLAST search will return two groups of relatives within the axial proteins:

  • FlgBCFGEK, the rod+hook+first linker protein, is one related group
  • FlgL (the 2nd linker) + FliC (flagellin) is the second related group

Figure 3 supports these already-known relationships, which are all supported by 24 or more hits, usually 30 or more hits.

However, the point of Liu & Ochman’s Figure 3is that not just the axial proteins, but all 24 proteins are connected by one or more homologies to other proteins. Just to make sure the authors really mean this, let’s examine relevant bits of the paper.

It is kind of strange to read through the Liu and Ochman paper – you can almost watch their thesis mutate from an interesting, if unoriginal, observation about axial proteins, to a sweeping, wildly unsupportable conclusion about all core flagellar proteins:

[Abstract] These results show that core components of the bacterial flagellum originated through the successive duplication and modification of a few, or perhaps even a single, precursor gene.

OK, here, maybe they just mean *some* of the core flagellar components came from a single common ancestor.

[End of the introduction] Our results show that flagellum [should be “the flagellum”, sic] originated very early, before the diversification of contemporary bacterial phyla, and evolved in a stepwise fashion through a series of gene duplication, loss and transfer events.

This is not wildly wrong as stated, although the phylogenetic conclusion about flagella being in the last common ancestor of bacteria is disputed, e.g. by Cavalier-Smith. Moving on:

Core Flagellar Proteins Arose Through the Duplication and Diversification of a Single Precursor.

That is pretty clear right there in the section heading: “Single Precursor.”

Core Flagellar Proteins Arose Through the Duplication and Diversification of a Single Precursor. When each of the 24 core flagellar proteins of E. coli are compared (via BLAST) to all proteins encoded in the E. coli genome, their best and often only hits are to other core flagellar proteins. This pattern indicates that the structural genes specifying the portion of flagellum residing outside of cytoplasmic membrane (i.e., the rod, hook, and filament) are paralogs and were derived from one another through duplications.

Here they are just talking about the E. coli K12 genome. We have already discussed the axial proteins, so you know this part is confirming conventional wisdom (connecting the FliC+FlgL group to the FlgBCFGEK group is a controversial addition, but that problem is small potatoes at the moment).

Let’s look at Figure 3 again.

The E. coli hits are the blue lines, and the matched E. coli proteins are the yellow boxes. I count 14 15 blue lines linking 10 yellow boxes, all axial proteins.

The paper continues on, to look for homologies outside of the flagellum, inside the E. coli K12 genome:

Aside from these matches to other core proteins, pairwise comparisons of these flagellar proteins to the >4,000 nonflagellar proteins encoded by the entire E. coli genome recovered cumulatively a total of only 24 hits that reached the same level of significance. […]

We will skip over some details which are vague and problematic for other reasons, but which are, again, small potatoes. The conclusion of their search of E. coli:

Thus, we conclude that despite their antiquity, the similarities among core proteins to one another are more common and, on average, stronger than to nonflagellar proteins.

It is none too clear, but I think what they are trying to say here is that with the E. coli K-12 genome, they got:

(a) 14 15 homologies found between 10 flagellar proteins (out of 24 total flagellar proteins)

(b) and 24 homologies found between ?? flagellar proteins (they don’t say how many) and 4,000 nonflagellar proteins

Because (a) is a higher proportion than (b), the authors say that “on average” the within-flagellum homologies are more common. OK I guess. This doesn’t establish anything about all the flagellar proteins evolving via internal duplications – if you’ve got external homologies then you have evidence that flagellar proteins could have once had an nonflagellar ancestors.

What about the other 40 genomes? The authors continue:

We repeated this analysis and compared the core gene set of each other flagellated bacterium to all proteins encoded in the corresponding genomes and among themselves, and we obtained a similar result, i.e., the best (and often the only) hits of the flagellar core genes were to other flagellar core genes.

This is just more of the axial protein homology detection.

However, by extending this analysis beyond E. coli, the similarity-relationships and links among several other core genes were resolved. For example, a highly significant match between fliM and fliN (that was not detected for E. coli homologs) was evident in 15 genomes from diverse bacterial subdivisions (Fig. 3).

The FliM-FliN homology has been well-known for quite a while. Apparently they don’t catch the well-known homology between the chemotaxis protein CheC and FliM because they don’t do a general BLAST search of a full database. (FliM has two main domains, homologous to CheC and FliN respectively.)

Cumulatively, each of the 24 core genes shows significant similarly to one or more of the other core genes (Fig. 3), a pattern that would result from their successive origination from one another by independent gene duplications and/or gene fusions.

See, I wasn’t making it up. They really are basing their all-from-one-gene conclusion on Figure 3.

Comparisons of the complete genome sequences of flagellated bacteria revealed that the flagellum is based on an ancestral set of 24 core genes for which homologs are present in genomes of all bacterial phyla. The most striking finding from our analysis is that these core genes originated from one another through a series of duplications, an inference based on the fact that they still retain significant sequence homology.

“[T]hese core genes originated from one another” – pretty clear what they meant.

Although sequence similarities among some of the rod and hook proteins were noted in early analyses (24), the degree of paralogy for the ancestral set of flagellar genes, and its implications for the origins of the bacterial flagellum, have gone unrecognized.

Um, right. That’s because the paralogy has not been detected by anyone else, except for the axial proteins and FliN-FliM. Figure 3 reports about 50 new homology matches (the grey lines plus a few blue lines) for 14 15 flagellar proteins. Authors and reviewers might have pondered profitably the question of why no one else was able to BLAST up this cornucopia of results before.

[Summary]

To ascertain the ancestry of the flagellar core genes, we searched initially for homologs of each gene within the E. coli genome, which has the highest proportion of functionally annotated genes. The resulting network, involving only 10 of the 24 core genes, provided a very conservative view of the relationships and paralogy among the core genes but showed that flagellar genes were derived largely from other flagellar genes with apparently little input from other coding sequences.

All this means is that within a single E. coli genome (K-12) they didn’t find much beyond the axial protein homologies.

Extending these analyses to include other genomes uncovered additional links among flagellar proteins and revealed that the entire set of core genes could be formed through the duplication and divergence of previously existing flagellar genes.

Figure 3 again. Yep, I guess they really do think this. “Entire set” is not ambiguous.

That the analysis of the E. coli did not resolve all of the links among core genes is not surprising given that these genes are ancient and have followed independent histories within bacterial lineages. It was originally hypothesized that biological pathways and structures might expand through the successive addition and modification of their preceding components (27). Although there is diminishing evidence that the recruitment of new enzymes into metabolic pathways occurs by this process (28), it is apparently the manner by which the bacterial flagellum arose.

In other words, the all-internal-duplications model has failed for metabolic pathways, and the cooption-from-diverse-sources model is now dominant – but we’ll resurrect the old discredited model for the flagellum!

[end of the conclusion paragraph]

As with the evolution of other complex structures and processes (29–32), we have shown the bacterial flagellum too originated from “so simple a beginning,” in this case, a single gene that underwent successive duplications and subsequent diversification during the early evolution of Bacteria.

This conclusion sentence is the 5th or 6th time the authors explicitly endorse the “all flagellar genes came from one” model. So let’s not have any more questions about what the authors meant to say.

Now, why am I skeptical? I posted some scattershot reasons on Monday. Some us PT posters are chewing on more technical issues. But to really get a visceral sense of the problem of claiming that all the flagellar genes are homologous, you just have to look at some structures. Here is FliI, the ATPase that powers the secretion of axial proteins like FliC:

(from Imada et al. 2007, PNAS)

Here is FliC, aka flagellin, which is supposed to be just two homology links away from FliI in the Liu/Ochman Figure 3:

(from Yonekura et al. 2003, Nature)

You can also compare the two proteins when assembled into multimers. FliI assembles into a homohexamer:

(from Imada et al. 2007, PNAS)

FliC assembles into the flagellar filament:

(from here)

For comparison, this is what two protein structures look like when they are homologous. These are the homologous domains in CheC (a chemotaxis protein in some flagella), and FliM (part of the flagellum base, which interacts with chemotaxis proteins to produce the switching in rotation direction). (FliM is basically two main domains, homologous to CheC + FliN. Liu & Ochman caught the FliN homology but apparently not the CheC one.)

(from Park et al. 2006, in PNAS)

You might have noticed that FliC and FliI do not exactly exhibit the kind of structural similarity shown by the homologous FliM and CheC.

I conclude that FliC and FliI are not homologous. Therefore not all core flagellar proteins are homologous. Therefore they didn’t come from a single ancestral ur-flagellar gene. Therefore the conclusion of Liu & Ochman is wrong. Case closed.

2 TrackBacks

As the discussion over the Liu-Ochman flagellum evolution paper continues, it is clear that I need to do a little more arguing to defend my position. Although some were convinced that skepticism was justified based the previous PT posts... Read More

A correction to the paper by Liu & Ochman, “Stepwise formation of the bacterial flagellar system,” was just published in PNAS. PT readers will recall that I and others had many problems with the methods and conclusions of this... Read More

63 Comments

I was in the “maybe there was a slight misunderstanding camp”, but I see that the flaws could go deeper. This is not, for the moment anyway, good work to use for ID bashing.

That’s okay for me, as I’m just a theorist – I’m not terribly worried about which specific mechanism it was; I just focus on the specifics of potential mechanisms. I’m happy to bash with the highly plausible existence of indirect mechanisms that they pretend don’t “really” count.

A couple of minor edits:

I count 15 blue lines connecting the 10 yellow boxes.

Here is FliC, aka flagellin, which is supposed to be just two homology links away from FliC in the Liu/Ochman Figure 3:

The second FliC should be FliI.

A very astute and incisive set of observations. Any chance of a bit of annotation on the structures, for those readers who don’t have experience in biochemistry? E.g. identification of beta-sheet and alpha-helices?

By the look of it, the FliC protein has loads of alpha-helix and no beta-sheet, which is rather different from the FliI protein. FliI seems to have several short alpha-helices, an anti-parallel beta-sheet, and maybe a little beta-strand.

Additionally, by my count, 18 of the homologies idenitfied in their figure 3 are supported by only 1 “hit” in 41 genomes, which is rather weak evidence on which to base such a sweeping conclusion.

We all know that protein structure and protein homology are notoriously difficult to correlate – elsewise, Pande wouldn’t have a gazillion PCs crunching the folding data. I, for one, would rather see the sequence data than the structural maps.

Hi Nick,

Thanks for the update – I look forward to seeing your reanalysis of their data and rebuttal of their interpretation in PNAS or another peer-reviewed venue.

PNAS is a fairly prestigious high profile journal to get published in, but back in the 1980’s I got a chuckle out of one of the professors on my committee (frankly, I didn’t notice him in the small departmental library) when I remarked to another grad student about passing the Proclamations of the National Academy.

I don’t know what the review process is today, but back then members could publish their own work or submit the work of others for publication. To get published you would submit your research to a member and they would have it reviewed and if you passed it got published. There were leaks in the system. That is about all I have to say.

Thanks – I fixed 14 and FliC/FliI mixup. Hard to count all them lines.

Nick,

I follow you through the problems with using BLAST to determine homology. That’s a tricky subject and I think we all await the day when our homology searches are based more on model-based methods and less on a quick similarity algorithm.

But you lose me at the protein structure bit, where your assessment is little more than a subjective “These look different to me so they can’t be homologous”. That’s not a rigourous argument, Nick, that’s an assertive opinion, and it suffers from the same logical problem as claiming that horse’s hooves and monkey’s feet can’t be homologous because they look different. You seem to be declaring that the external morphology of a molecule is more important in homology assessment than the sequence of amino acids within the molecule, and while that may or may not be true it is ad hoc reasoning to declare the one way is the correct way to do it.

In the meantime, what are we to make of the observation that there are no obvious homologies from some of the flagellar proteins to anything outside the flagellum? Your favored scenario implies co-option of proteins from other systems, but you seem locked in a logical spiral where your evidence for external homology comes from within the story that you wish to tell, not from any outside evidence of homology. This isn’t to say that I’ve got anything against your scenario- I find it quite elegant- but your supporting evidence is at least as scant as that of Riu and Ochmann.

I think Nick’s point is that the structures are very divergent, so much that existing models of flagella evolution do not propose that all flagellar proteins evolved from a common ancestor. The model of Liu and Ochman goes against current wisdom, and lacks strong evidence to back it up. Finding BLAST hits in a large database does not guarantee that those hits will be evolutionary significant. I haven’t had time to look at the supplementary material, but several people I trust say that some of these homologies are artifacts.

Reed,

Having an internally consistent model for the evolution of the flagellum is not the same thing as having external corroborating evidence for that model. The rejection of putative homologies on the grounds that they don’t fit with the model is precisely backwards for how one ought to do science, yet that is what you and Nick are suggesting. Shall we be critical of all data that don’t fit Nick’s scenario, or shall we be critical of Nick’s scenario when the data don’t appear to fit it?

There are certainly issues with using BLAST to assess homology, and perhaps those criticisms are well-founded, in which case my point is moot. But I’m not about to accept a blogosphere debate on the topic over the normal peer-review process, however flawed it may be.

Hi Nick,

I’m no biologist, but this topic is interesting to me (and, I assume, many other non-experts), and I would like to understand your critique in more detail. As others in this thread have pointed out, your dismissal of the proposed homologies isn’t really argued for in detail, and it’s hard for someone with no experience in “reading” protein structures to understand the significance of the images you present.

I take it from your categorical “case closed” that you find the structures conclusive. Could you help those of us who want to understand the issue but don’t have the necessary training by pointing out some of the features that lead you to this conclusion? Are there particular properties that rule out homology, or are the structures simply so different that any biologist would immediately (and correctly) decide that there is no chance that the proteins are homologous?

Also, I’m wondering whether there are any structure-based similarity measurements that could be used to quantify the similarity — somehow “they look different to me, and I’m an expert” strikes me as, well, unscientific..

Myrmecos,

You have it wrong. The paper is raising issues because it lacks convincing evidence for its novel homologies. A single BLAST hit out of what—over 100,000 comparisons—does not suggest that the hit is not an artifact. The gray lines are the blue lines don’t seem to be comparable.

There is much more discussion going on in email than you have seen on PT. It is my understanding that those details will be published on PT.

“There are certainly issues with using BLAST to assess homology, and perhaps those criticisms are well-founded,…”

You could say that indeed, and that’s what is being said here. Nick is not the only one looking at this. In fact he is communicating with others who are looking closely at the paper, and under the circumstances many more people must be examining it. Have you noticed any rebuttal?

“ … in which case my point is moot. But I’m not about to accept a blogosphere debate on the topic over the normal peer-review process, however flawed it may be.”

There will surely be more flagellum papers. Meanwhile I predict that the blog approach will turn out to be a constructive lesson, or lessons, for many people including some who don’t, and some who do, read the journals. You may like it better in time. I agree though that this was a bit startling, and different reactions are not surprising.

Nick,

It seems the topic is too complex for a mere letter to the editor. It might be more appropriate to write a rebuttal paper. What you need is statistically significant evidence for homology of some of the genes to genes other than those proposed in the paper. Even just a few obvious examples should suffice. I still think it would be best to get some well known names in the field as co-authors.

Pete is right, we should learn some valuable lessons here about the role of the internet in such debates. Once this issue is resolved, it should serve as a shining example of the self-correcting nature of science. The most important thing to remember is that ‘poof’ is no longer on the table.

Daniel Morgan Wrote:

We all know that protein structure and protein homology are notoriously difficult to correlate — elsewise, Pande wouldn’t have a gazillion PCs crunching the folding data. I, for one, would rather see the sequence data than the structural maps

It isn’t so much that protein structural homology and protein sequence homology are hard to correlate. The protein folding problem is usually approaching it from an ab initio approach (and this is slightly off topic but I work with protein sequence/structure homology quite extensively). which is why it takes so much computational power. Homology modeling on the other hand, which relies explicitly on the correlation between sequence homology and structural homology (because structural homology is retained much longer than sequence homology) is quite robust.

However I too would like to see the blast results themselves and the pairwise alignments generated. Their scores are fairly significant for detecting homology but artifacts do occur. When I was reading the paper I would have been fairly comfortable with a declaration that the flagellum arose through the successive duplication of several ancestral proteins and I think their data would have supported that position.

Nick, I agree with your conclusion on what Liu and Ochman are asserting. I side with several other commenters that your argument from structure is weaker than it could be. I know the tertiary structure is notoriously difficult to derive straight from the genetic sequence. Your argument seems to be that all homologies in sequence space should be obvious from arbitrary rotations of 3D structures. I can’t buy it. Park, et al. 2006 wants to convince us of the homology of FliM and CheC, and rotates and colors the model to help make that argument. You take two proteins that not homologous to each other, but rather to an intermediate, which are modeled and rotated arbitrarily with respect to each other, using different graphical systems, and expect the reader to conclude based on appearance that you have invalidated Liu and Ochman’s argument.

Sorry, you lose. If I squint hard enough, I can see the tail of FliC as an extension of the lower part of FliI, and the assembled FliC as a stacked set of homo-n-mers.

Your argument would be more convincing, though not dispositive, if you could show the intermediate protein’s teriary structure, show all three as ribbon diagrams of the same orientation and coloring, and if you could show that the changes in sequence should not be expected to result in the corresponding changes in tertiary structure. My naive expectation is that the intermediate has a tail of size intermediate between FliC and FliI. You’d go a long way to convincing me if you could show my expectation is wrong.

To the point that some of the lines are supported by only one hit out of 41, I would counter that (as you point out) there is some conservatism in e value. A looser e value might support more hits, but the fact that the authors stuck with a conservative value argues in favor of an attempt to do careful work and following the data to a conclusion, rather than a BLAST fishing expedition.

Cripes, how much different could those structures get? At the very least it would take a detailed argument to conclude homology, not 2 hits out of 41 genomes (which is the only thing that connects FliI to FliD, which is connected to FliC).

Typically the rule is:

Sequence similarity that is weak, but statistically strong enough to conclude homology, predicts almost superimposable structures in the homologous domains, e.g. like FliM and CheC.

Here’s another argument for people. FliI is an ATPase. It is highly conserved, with well-known homologous in – literally – hundreds of different relatives. The closest relatives in BLAST and PSI-BLAST searches go something like this: FliI, F1-ATPase, RecA, PilT (from Type 4 Secretion), then the whole superfamily of AAA ATPases, and then even more. Heck, FliI is more closely related to eukaryotic dynein than it is to FliC or FliD. At least FliI and dynein both form similar hexameric superstructures that use ATP (with sequence-homologous domains) to effect conformation change, and at least this hit can be retrieved on PSI-BLAST (many of the Liu-Ochman claims cannot…we have tried).

For ATPase homologs see: Iyer LM, Leipe DD, Koonin EV, Aravind L. “Evolutionary history and higher order classification of AAA+ ATPases.” J Struct Biol. 2004 Apr-May;146(1-2):11-31.

Nick,

Have you considered a phylogenetic analysis of the appropriate genes? The homology hits and conserved structure approaches are not really the same thing as a cladistic analysis. Perhaps showing that some of the genes were more closely related to other genes than the ones proposed in the paper would help the argument. You could determine the degree of confidence in the tree and determine how many more steps it would take to force the topology into that proposed in the paper. I’m not sure how difficult this would be, since alignment issues might complicate things. Still, if you have sequences for the candidate ancestral genes it might be worth a try.

Nick Wrote:

Cripes, how much different could those structures get? At the very least it would take a detailed argument to conclude homology, not 2 hits out of 41 genomes (which is the only thing that connects FliI to FliD, which is connected to FliC).

Do I hear an argument from incredulity? I’m not clear why you think it would take a detailed argument to prove homology, when a handwaving argument was enough to disprove it in your post.

If Liu and Ochman have built a cladogram that depends upon shaky homologies in your opinion, just say so. You start off by calling their pairwise homology hits “significant”, so I think you’ve undercut that argument. If they had published Fig 3 with e=0.01, gotten only 2 out of 41 pairwise homologies, and then made strong claims, you’d be home free. But at e=0.0001 it looks like you’ve got to work a little harder to convince me.

Also let’s remember that proteins can have similar three-dimensional structures and still have little to no primary sequence similarity. For example mandelate racemase and muconate lactonizing enzyme from Pseudomonas ovalis have virtually identical 3-D structures even though they only share 26% sequence homology. L-aspartate amino transferase and D-amino acid aminotransferase (PDB 2aat, 1daa) have similar tertiary structures even though they have very little in common, sequence-wise. Likewise tryptophan synthase (PDB 1ttp) and galactonate dehydratase have very similar alpha/beta barrel domains even though there is no sequence homology between the two proteins.

David,

A significant hit in a BLAST search is not the same as a significant relationship evolutionarily. Unrelated proteins can have parts that are similar due to convergent evolution.

Nick Matzke Wrote:

Because (a) is a higher proportion than (b), the authors say that “on average” the within-flagellum homologies are more common. OK I guess. This doesn’t establish anything about all the flagellar proteins evolving via internal duplications — if you’ve got external homologies then you have evidence that flagellar proteins could have once had an nonflagellar ancestors.

This poor non-biologist can’t follow far into the wondrous land of structural vs sequence homology. But this suggestion that the grouping may be an artifact of the analysis seems easy pickings.

Nick Matzke Wrote:

Here’s another argument for people. FliI is an ATPase. It is highly conserved, with well-known homologous in — literally — hundreds of different relatives.

There you go.

I can believe, you NIck. PNAS has been publishing lots of really crappy stuff.

The use of other genomes to identify related sequences is entirely reasonable - for the same reason, it is helpful to have “dense taxon sampling” when resolving phylogenetic relationships. Furthermore, distantly related sequences can help resolve taxonomic relationships between closely related species. This is a fact.

Nick, if a single lineage within E. coli is fast evolving, but more slowly evolving in another species, then it is perfectly reasonable to ID relatives by BLAST in other species that are not found in the same genome.

Furthermore, as others have commented, the structure homology assertions have no logical extra weight than sequence homology assertions. This is the sad truth about comparing distantly related sequences

I have to say you are completely wrong here.

Please sir, may I kindly backpedal? Somehow I got carried away with an argument that I thought you were making, but weren’t.

My bad.

I also feel compelled to point out that the second author and the editor of the paper are very, very good scientists who publish some damn good work. I’ll admit I should go back over the paper a little more critically, and I don’t agree with the assertion that there was one ancestral sequence as opposed to a small group of ancestral sequences. There may be something off, flagellum evolutionary relationships isn’t exactly my expertise, but I’m not ready to hang the authors out to dry just yet like some seem to want to do. Critiquing the paper is all well and good but if in my opinion if you disagree vehemently you work up a paper of your own and publish a rebuttal, or you write something as a commentary.

“A significant hit in a BLAST search is not the same as a significant relationship evolutionarily. Unrelated proteins can have parts that are similar due to convergent evolution.”

Reed,

Thanks for responding. That is exactly the point I was trying to make. This is why BLAST searches can yield spurious results. The figures from the paper are not really a phylogenetic analysis.

What I had in mind was something like what Morris Goodman did for the globin gene story. All the genes were derived from one ancestral sequence. He demonstrated their relationships and estimated divergence dates with a phylogenetic analysis. If any of the genes were not derived by duplication from the ancestral sequence, they would not fit into the same clade as the other genes. If you included other possible ancestral genes in the analysis, the genes derived from them should group with them rather than with the other clade.

Of course this problem is a little more complex. The divergence dates are longer. There are bound to be problems with alignment, long branch attraction, identification of ancestral sequences, etc. Still it might be worth a try.

Sorry if I’m being too simplistic or if there is something I’m missing. Just a suggestion.

David Stanton,

Note that my comment was directed to the other David.

Reed,

Sorry. What’s all this about Presidential Aids? Never mind.

The idea that a phylogenetic analysis somehow gives information about homology but a BLAST hit doesn’t isn’t correct. If I can find a protein by BLAST, then I can align those proteins. If I can align those proteins, I can build a tree. By building a tree one is already, by definition, assuming homology.

Now, I will agree that a phylogenetic tree can give information about degrees of relatedness. But the definition of homologous is that the sequences are “descendants of a common ancestor.” The only way two proteins could not be homologous at some level is if at least one of them evolved de novo out of dna sequence that was not derived in any way from the ancestor of the other protein. Like an intron evolving into an exon…

Douglas:

I was under the impression they were most likely using blastall as opposed to blast2seq for their studies although I could be mistaken. If they used bl2seq I’m sure that they would have adjusted accordingly. I don’t generally use bl2seq myself as I’m usually doing larger scale analysis.

Hahaha nevermind, I’m a complete idiot. Of course they used Bl2seq for their pairwise comparisons. So yes the point stands that e-value correction is required here. Thanks for reminding me Douglas

Here is what we mean by methodological problems and database size issues.

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