12 December 2012
Dear Dr. Balloux,
We would like to present our revised manuscript, "The fester locus in Botryllus schlosseri
experiences selection" for your consideration. We have addressed all the concerns raised by
the two referees, particularly regarding the discussion. We sincerely hope this revision meets
the reviewers' expectations.
In particular, Reviewer 1 asked us to "Please include a few sentences in the discussion section
commenting on the finding of either purifying or directional selection at fester in the context of
the evidence for both costs and benefits of fusion in ascidians." We have done so, and have
included all the literature listed by the reviewer (please see Lines 393-399 and 405-412 in the
revised manuscript).
Reviewer 2 said, "The writing is very loose in parts, especially the discussion. There needs to be
a coherent argument being set out." We have revised both the introduction and discussion to
focus on a coherent argument. Reviewer 2 also said, "Discussion starts very abruptly, does not
lead into building a main case." We have reorganized the discussion so that it starts by leading
into the main case (whether or not fester is experiencing selection, and if so, what type of
selection).
We have uploaded the revised manuscript, along with a version highlighting all the changes
made to the original manuscript. Our response to all the reviewers' comments is reproduced
below.
Thank you for your attention to our manuscript,
Marie Nydam, Ph.D.
Anthony De Tomaso, Ph.D.
Authors' Responses to Reviewers' Comments
Reviewer 1 Comment 1
At the very least, the authors need to discuss the findings of their study in the context of the
suite of papers that suggest that fusion is an important component of ascidian life history,
particularly given their finding of purifying or directional selection on a locus association with
fusion.
Please include a few sentences in the discussion section commenting on the finding of either
purifying or directional selection at fester in the context of the evidence for both costs and
benefits of fusion in ascidians.
Author’s Response to Reviewer 1 Comment 1
We appreciate the Reviewer’s comments and the associated literature citations. We have
added a section based on the reviewer’s suggestions, which includes all the literature suggested
by the reviewer (please see Lines 393-399 and 405-412).
Reviewer 1 Comment 2
A brief discussion of the finding of purifying or directional selection in fester in comparison with
the recent findings of hyperdiversity at the allorecognition loci in hydractinia, (Gloria-Soria et
al., 2012; Nicotra et al., 2009; Rosengarten et al., 2011) would also greatly enhance the impact
of this study.
Author’s Response to Reviewer 1 Comment 2
We appreciate the Reviewer’s comments and the associated literature citations, including the
new work by Gloria-Soria et al. We agree that the work on Hydractinia alr2 is a useful addition
to the discussion of the current manuscript, especially in light of the conclusions that GloriaSoria et al. makes regarding the nature of selection on alr2. We have brought Gloria-Soria et al.
into the discussion (please see Lines 400-404).
Reviewer 2 Comment 1
Although this revised submission is better prepared than the original, most of my previous
concerns have not been addressed. Frustratingly, there does not seem to be any response to
reviewers letter in the documents associated with this revision. I will assume that this is the
fault of the journal and not the authors but will nonetheless evaluate the revision against my
original comments.
Authors’ Response to Reviewer 2 Comment 1
We apologize for the confusion. We wrote a 15 page “Response to Reviewers” document and
uploaded it with our first revision, but it seems that you did not receive it. We sincerely hope
that the current document reaches you.
Reviewer 2 Comment 2
All results based on housekeeping loci are buried in the supplemental data.
Authors’ Response to Reviewer 2 Comment 2
We have moved the summary statistics for the housekeeping genes to the main text of the
paper (Table 1).
Reviewer 2 Comment 3
Moreover, by having 10 additional loci, it is possible to statistically compare summary statistics
from fester (diversity measures, Tajima’s D, etc) against statistics from the other loci, thus
effectively controlling for demographic changes affecting the whole genome; this would greatly
strengthen the inference of this study. For instance, a series of plots for each statistic showing
the spread of values among loci (and highlighting fester) would be illuminating and quickly
convey the results. The key thing would be to *statistically* show that fester differ from other
loci.
Authors' Response to Reviewer 2 Comment 3
We have added four figures that explicitly compare Tajima's D values, Fu and Li's D* values, Fu
and Li's F* values, and AMOVA statistics between the housekeeping genes and fester. Please
see Figures 3-6 in the revised manuscript. For all of the statistics, the fester values and the
housekeeping values are non-overlapping. These figures provide incontrovertible evidence that
fester differs from other loci, and we thank the reviewer for the suggestion. We have modified
the Results and Discussion to highlight these new Figures.
Reviewer 2 Comment 4
Within fester, why were tests of selection not performed on all alleles together? This would
corroborate your contention of balancing selection (most likely).
Authors' Response to Reviewer 2 Comment 4
We would like to refer the reviewer to the "Comparison of variation among fester allele types"
section of the Methods: For all population-level analyses, we analyze each allele type separately
because each has a separate evolutionary history, and because including divergent alleles in the
same data set could create artifacts (e.g. Wahlund effect). In response to the reviewer's
comment, Tajima's D and Fu and Li's D* and F* could show positive values for several different
reasons, including population subdivision with even sampling across subpopulations. By testing
each allele type separately, we minimize the risk of such artifacts.
Reviewer 2 Comment 5
It is also odd, that no outgroup was sequenced – adding outgroup sequences would enable far
more sensitive (and nuanced) metrics of selection to be applied such as McDonald-Kreitman
and HKA tests.
Authors' Response to Reviewer 2 Comment 5
We wholeheartedly agree that an outgroup sequence would be very useful. However, there are
several reasons why obtaining such a sequence is not possible at this point.
First, FuHC/fester has only been described from B. schlosseri. Although many other botryllid
species exhibit allorecognition, it is not known whether they use FuHC/fester or a different
system or systems. If other species do have FuHC/fester, these genes could be substantially
divergent from B. schlosseri FuHC/fester. Attempts to amplify fester from two other botryllid
species (B. violaceus and B. diegensis) using B. schlosseri fester primers have not been
successful. Degenerate primers are not possible, given that we only have fester sequence from
a single species.
A related problem is the phylogenetic relationship of B. schlosseri to the other botryllids. If we
knew the closest relatives of B. schlosseri, we might have more success amplifying fester-like
genes from these relatives using B. schlosseri fester primers. We have tried to place B.
schlosseri using the only widespread botryllid sequence on GenBank (18S), but the position of
this species in the botryllid phylogeny is unstable (i.e. the position varies depending on which
18S sequence is used). We would eventually like to create a botryllid phylogeny, but many of
the species are difficult to access. Genome-wide sequencing has only been done for one other
botryllid (B. primigenus), and we asked the researcher in charge of these unpublished
sequences if he would search for fester-like genes. He did so, and did not find any significant
matches to B. schlosseri fester.
Reviewer 2 Comment 6
There has been no attempt to statistically demonstrate selection, that is to show that shared
demography across loci can be excluded as the explanation of strange results for fester. Why
not use the tests of selection previously suggested?
Authors’ Response to Reviewer 2 Comment 6
We would very much like to implement McDonald-Kreitman and HKA tests. However, these
tests require sequences from a closely related species. Please see our response to Reviewer 2
Comment 5 on why we were not able to obtain fester sequences from an appropriate
congener.
Reviewer 2 Comment 7
The term “selection” is used frequently, but it is often unclear what nature of selection is being
referred to. It is especially important to lay out the expectations regarding fester evolution in
the introduction. For instance, balancing selection seems like a reasonable hypothesis given
what we know about other recognition loci. The specific expectations for balancing selection,
however, could be subtle. For instance, one might expect to find balancing selection when
looking across all alleles (in combination) but directional selection within each cluster of alleles.
OR, is the BS signal expected within clusters as well? What is the predicted underlying process
driving selection and how should this be manifest at the molecular level?
Authors’ Response to Reviewer 2 Comment 7
We have completely revised the Introduction to reflect the reviewer’s requests. Please see
Lines 142-160.
Reviewer 2 Comment 8
The writing is very loose in parts, especially the discussion. There needs to be a coherent
argument being set out.
Authors’ Response to Reviewer 2 Comment 8
We have completely revised our discussion to make our argument more coherent (please see
Lines 310-412). We sincerely hope this revision meets the reviewer's expectations. Note: Lines
364-372 were added at the request of a reviewer.
Reviewer 2 Comment 9
Line 52, Line 313 – What type of selection?
Authors’ Response to Reviewer 2 Comment 9
We have fixed Line 52 to make it clearer. We have clarified the nature of selection in Line 313.
Reviewer 2 Comment 10
End of introduction – vague. What are the predictions, expectations, hypotheses?
Authors’ Response to Reviewer 2 Comment 10
We have completely revised the end of the introduction, and removed the paragraph to which
the reviewer refers.
Reviewer 2 Comment 11
Results – The format of having no methods in the main text is awkward and a challenge for any
author. Yet, the results as written need to go further in providing rationale/purpose for the
different methods.
Authors’ Response to Reviewer 2 Comment 11
We added information in several places throughout the Results to explain the purpose behind
our Methods. Please see Lines 172-173, 180-181 and 207-209. We have also revised the
Introduction to include explicit rationale and predictions for each of our Methods (please see
Lines 142-160.
Reviewer 2 Comment 12
Results: The housekeeping genes should be listed by name.
Authors’ Response to Reviewer 2 Comment 12
We have added a list of housekeeping genes to the Results section (please see Lines 227-230).
Reviewer 2 Comment 13
The results section also had switching back and forth between present and past tense. Be
consistent.
Authors’ Response to Reviewer 2 Comment 13
We have changed everything in the Results to the present tense. Where we discuss Methods in
the Results section, it is necessary to keep these sentences in the past tense. These sentences
are reproduced here. “Colonies were collected from floating docks in each of 6 populations in
2009 and 2010.” “We sampled 22 colonies with A-type alleles from the East Coast, and 18
colonies with A-type alleles from the West Coast.” “To further compare housekeeping genes and
fester, we calculated mean Tajima's D, Fu and Li's D*, and Fu and Li's F* values (across all six
populations) for housekeeping genes.”
Reviewer 2 Comment 14
Results/Methods – if there is recombination (and there is), then there is no real gene tree.
“Clades” may not be monophyletic. Furthermore, genealogical based methods of testing for
selection may be affected by recombination. This is all the more reason to consider tests of
selection such as McDonald-Kreitman and HKA tests.
Authors’ Response to Reviewer 2 Comment 14
We agree that genealogical based methods of testing for selection may be affected by
recombination. For Tajima’s D and Fu and Li’s D* and F* statistics, we estimated the intragenic
recombination in DnaSP. This recombination rate was then included in the input parameters
when we calculated Tajima’s D, Fu and Li’s D* and F*. We explained this in the Methods
section entitled “Tests of selection: polymorphism statistics”. To calculate ω (dN/dS), we used
omegaMap, which is specifically designed to calculate ω in the presence of recombination. We
explained this in the Methods section entitled “Tests of selection: ω statistics”.
Reviewer 2 Comment 15
AMOVAs are not tests of selection. They can inform the nature of selection.
Authors’ Response to Reviewer 2 Comment 15
Where we discuss AMOVA and FST analyses we have changed the headings from “Tests of
selection…” to “Selection inference”.
Reviewer 2 Comment 16
Discussion starts very abruptly, does not lead into building a main case.
Authors’ Response to Reviewer 2 Comment 16
We have completely reorganized the discussion so that it starts by leading into the main case
(whether or not fester is experiencing selection, and if so, what type of selection). We sincerely
hope this revision meets the reviewer's expectations.
Reviewer 2 Comment 17
Line 555 – A Wahlund effect refers to patterns of linkage disequilibria, not divergence among
alleles.
Authors’ Response to Reviewer 2 Comment 17
We removed the phrase “Wahlund effect”.
Reviewer 2 Comment 18
Figs 3-6 are a big improvement over the previous graphics. However, each locus should be
identifiable by name.
Authors’ Response to Reviewer 2 Comment 18
We attempted to label each bar on each figure, but the figures were unreadable. So we
identified each locus (in order) in each Figure Legend (Figures 3-6).
Reviewer 2 Comment 19
Fig 4 & 5 are identical.
Authors’ Response to Reviewer 2 Comment 19
We apologize if we accidentally uploaded the same figure twice. Figure 4 and 5 do look very
similar, but should not be exactly the same. The y-axes have different scales, for instance. In
Figure 4 (Tajima’s D), the sixth and seventh bars from the left should be barely visible because
they are at zero. This is not the case with Figure 5 (Fu and Li’s D*) – the sixth and seventh bars
from the left are both well above zero.