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Re: Mesozoic roots of parrots and passerine birds



Hello everyone,

I am glad that you are interested in our recent paper in Nature
Communications. While reading your discussion on http://dml.cmnh.org, I felt
very tempted to add a few comments. I hope I can answer a few questions by
doing that now. :-)


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Remarks on the name Eufalconimorphae (a reply to
http://dml.cmnh.org/2011Aug/msg00401.html):

I admit that the term Eufalconimorphae is, on the first thought, a slightly
confusing name for parrots + passerines + falcons and implies that it is
nested within a clade named "Falconimorphae" - the latter name is nowhere to
be found in our paper. Here are a few reasons why this term nevertheless makes
sense (to me, at least):
- the term "true Falconimorphae" indeed indicates that the "true falcons" do
not group together with the other "traditional Falconiformes" (Accipitridae,
Pandionidae, Sagittariidae).
- it leaves "taxonomic space" for a re-definition of the term Falconimorphae
(it was used in the Livezey & Zusi 2007 taxonomy for Cathartidae +
"traditional Falconiformes" + Strigiformes), such as the clade
Eufalconimorphae + Cariamidae. Personally, I wouldn't yet dare to name this
clade (even though also Ericson et al. 2006 and Hackett et al. 2008 recover
this clade with moderate support), as our study only revealed a support of 2
retroposon insertions (= nice, but not statistically significant). Maybe
upcoming sequence-based or retroposon-based studys will find more support for
Eufalconimorphae + Cariamidae? Anyways, I look forward to reading other
independent studies on this issue.
- Psittacopasserae being part of the taxon Eufalconimorphae acknowledges the
possibility that (as most of the old "landbird" lineages are raptorial,
including the putative sister taxon of Eufalconimorphae) parrots and
passerines could have evolved from a raptorial ancestor. This coincides with
the observations by Mayr (2010) on a putative stem group Psittaciformes fossil
exhibiting a raptor-like beak and large processus supraorbitales.

By the way, the support for Eufalconimorphae was among the strongest in our
analysis (7 retroposon insertions), so I am quite positive that this grouping
is not an artefact.


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Remarks on the phylogenetic affinities of mousebirds:

What do you think about the hypothesis of mousebirds being the sister taxon of
all remaining "landbirds"? The support is only moderate (2 retroposons), but
appears to be stronger than the low resolution in Hackett et al. (2008)
placing mousebirds near Strigiformes. At least, it's something to be tested by
future studies. If this grouping is "true", I wonder how the ancestor of
"landbirds" might have looked like?


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Remarks on methodological doubts (a reply to
http://dml.cmnh.org/2011Aug/msg00400.html):

"Same problem as with transposons: indels were once believed to be entirely
nonhomoplasious, but they are apparently anything but that."
I agree that indels, especially the very small ones, are quite prone to
homoplasy. But please note that retroposon insertions are not indels - they
show a clear polarization of character states (ancestral: empty target site;
derived: a retroposon insertion flanked by a target site duplication) and
usually are much larger than indels (the ones in our study are about 200-600
bp). Hence, the occurrence of homoplasy (i.e., precise deletion of the
retroposon insertion including one of the duplicated target sites; or parallel
insertion of a very similar retroposon in the same target site + same
truncation + same orientation) is extremely low (= virtually homoplasy-free).
Although there are many vertebrate genomes sequenced now (making it easy to
examine the insertion sites of retroposons), there is, to my knowledge, no
evidence that the retroposons studied in our paper (CR1 subfamily of LINEs;
LTRs of endogenous retroviruses) insert at special sites in the genome. Please
note that in contrast to retroposons, some DNA transposons have target site
preferences (which is hard to study, because unlike retroposons, DNA
transposons don't stay in the same place once inserted), and thus are not used
in phylogenetic studies.
Already 10 years ago, to consider the fact that homoplasy is rare, but
possible in retroposon insertions, Waddell et al. (2001) have proposed a
statistical framework (>3 markers for a clade are statistically significant,
if there is no refuting signal). Finding 1 or 2 retroposons for one clade is
thus nice, but not sufficient for claiming that this grouping is not an
artefact.

"But verificationism hasn't been an acceptable scientific methodology 100
years ago! You need to look for data which REFUTE your hypothesis, and if you
can find none, THEN you're cool."
Well, I find the term "verificationism" wonderful, but maybe there's the
chance to convince you that this term does not apply to our study (or to the
use of retroposons in phylogenetics, in general). Let me briefly summarize the
approach and what that means in terms of our data set:
1. Computationally extract retroposons and flanking sequences from a sequenced
genome (in our case: zebra finch and chicken, and a low amount of emu
sequences; in total more than 200,000 retroposon insertions).
2. Select those that can be used for cross-species comparison (i.e., PCR
amplification and sequencing). This was the case for 206 retroposon
insertions, the majority of them were present in the zebra finch and absent in
chicken and emu - hence, they were retroposon marker candidates for resolving
the neoavian radiation.
3. Obtain sequences from all other birds in the taxon sampling via PCR
amplification and sequencing (this was possible for 51 marker candidates).
Some of the 206 marker candidates didn't make it, obviously - but that's not
surprising, given the "deep divergences" that were investigated.
4. Align sequences for each marker candidate and check which species exhibit
the respective retroposon insertion at an orthologous locus (coded as "+" in
our character matrix) or the ancestral state (the absence situation, coded as
"-" in our character matrix). Note that this can only be done after carefully
checking the alignments for retroposon boundaries and (most importantly) the
integration site (including target site duplication). Consequently, the
character state "-" means that the integration site is empty (if the
integration site was absent, i.e., due to a deletion, the character state was
coded as "d", which equals "missing data")
5. Construct a cladogram based on the character matrix of the 51
presence/absence patterns to see where the markers "sit" on the tree. To test
the strength of the retroposon support for eachbranch, conduct Waddell et
al.'s likelihood-ratio test (3 or more unconflicting retroposon markers for
one branch constitute statistically significant evidence). 47 of the 51
markers yielded a 100% congruent pattern (= the resultant tree topology): 3
retroposon insertions unite Psittacopasserae, 7 (!) unite Eufalconimorphae
(note that none of them are J-class retroposons aka CR1-J; instead, they are
endogenous retrovirus-derived LTRs), etc..
Interestingly, 4 of the 51 markers showed quite strange patterns putatively
caused by incomplete lineage sorting (these are the 4 J-class retroposons of
Supplementary Figure S2) - they are incongruent among each other and some of
them also with the markers for "landbirds" (clade G), "landbirds" without
mousebirds (clade H), and seriemas + Eufalconimorphae (clade I).
But note, NONE of them are incongruent with the 7 retroposon markers for
Eufalconimorphae and the 3 retroposon markers for Psittacopasserae (that's
supposed to be the main take-home message of Supplementary Figure S2).
Additionally, by doing the above described methodological procedure, one would
expect to find refuting signal (rejecting the Psittacopasserae and
Eufalconimorphae hypotheses, e.g., passerines + woodpeckers et al. or
passerines + cuckoos) with the same amount of chance, but this was not the
case.

To sum up, the earliest part of the neoavian radiation appears (with the
current amount of data) irresolvable, the middle part ("landbirds" and some
internal relationships) shows some congruent retroposon presence/absence
patterns, but also some incongruences (when considering the retroposons that
inserted in the earliest part of the radiation), and finally, the "youngest"
part of the radiation appears to be resolvable without conflicts
(Psittacopasserae, Eufalconimorphae). It's a start, and I look forward to
seeing the results of whole-genome/transcriptome comparisons on that issue.


Thanks for your patience in reading this. I'm glad to answer any questions, so
please feel free to contact me.

Best regards,
Alex


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References:
- Ericson, P. G. P. et al. Diversification of Neoaves: integration of
molecular sequence data and fossils. Biol. Lett. 2, 543–547 (2006).
- Hackett, S. J. et al. A phylogenomic study of birds reveals their
evolutionary history. Science 320, 1763–1768 (2008).
- Livezey, B. C. & Zusi, R. L. Higher-order phylogeny of modern birds
(Theropoda, Aves: Neornithes) based on comparative anatomy. II. Analysis and
discussion. Zool. J. Linn. Soc. 149, 1–95 (2007).
- Mayr, G. Well-preserved new skeleton of the Middle Eocene Messelastur
substantiates sister group relationship between Messelasturidae and
Halcyornithidae (Aves, ?Pan-Psittaciformes) Journal of Systematic
Palaeontology 9, 159-171 (2011).
- Waddell, P. J., Kishino, H. & Ota, R. A phylogenetic foundation for
comparative mammalian genomics. Genome Inform. 12, 141–154 (2001).