Peer review of: Interspecific introgression mediates adaptation to whole genome duplication (2019)

Reviewer #1 (Remarks to the Author):  

Review of ‘Interspecific introgression mediates adaptation to whole genome duplication’  

Marburger at el. describes a population genomic study in which they test the hypothesis that adaptive 
gene flow (of genes adapted to increase meiotic stability and/or reduce meiotic crossovers) from one 
species to another mediates escape from extinction following WGD. In particular, the authors show 
that specific pre-adapted alleles ‘donated’ from tetraploid Arabidopsis arenosa to tetraploid 
Arabidopsis lyrata (which has undergone a WGD more recently than arenosa) probably rescued the 
nascent A. lyrata tetraploids from early extinction.  

I have read this paper with great interest, but have the following comments.  

The authors mention several times that the pre-adapted alleles donated from A. arenosa to A. lyrata 
are responsible for rescuing A. lyrata from extinction. However, to this reviewer it is unclear whether 
tetraploid A. lyrata would indeed have gone extinct without adaptive introgression of alleles from A. 
arenosa or just adapt to WGD more slowly? Is there any way to ‘proof’ that, if not having obtained 
these adapted alleles by gene flow, the new tetraploids would have gone extinct? Is this suggested by 
the paragraph ‘stabilisation of lyrata meiosis following WGD’, where the authors mention that meiotic 
stability is segregating within tetraploid populations? This is not entirely clear to me. I have the feeling 
the authors might be more careful in phrasing this. I think the observation that pre-adapted genes 
have been obtained by gene flow is very interesting and for sure are a strong indication that these 
helped A. lyrata coping with the WGD (as has been shown previously for A. arenosa), but this is not 
the same as assuming that otherwise tetraploid A. lyrata should have gone extinct for sure. If true, 
then, we also need to assume that A. arenosa would have received its ‘adapted’ genes from 
somewhere else (or it too might have gone extinct ..).  

The authors state that ‘… selective sweep signatures directly overlapping eight loci that interact during 
prophase I..’. I assume that it is not the genetic loci that directly interact but rather their gene 
products?  

The authors state that they ‘… estimated the age of WGDs at 81k generations for lyrata (~160,000 
years)’. In this respect, I think the following papers could be cited as well (Hohmann and Koch, 2017; 
Novikova et al., 2018).  

The authors state that interspecific gene flow from tetraploid arenosa to tetraploid lyrata (0.0998 
alleles/generation) and lyrata to arenosa are at the same level (0.1001 alleles/generation)”  
How come arenosa->lyrata flow is lower than lyrata->arenosa? Admixture patterns and Dfoil results 
suggest the opposite, or am I missing something here?  

The authors experimentally show that meiotic stability is varying from population to population (Table 
S3), but they fail to show if it depends on the presence of particular sets of adaptive alleles (from 
Table 2, for example) or admixture assignments. Is this something that could be looked at?  

In general, I think it would be nice if the authors could briefly explain the population statistics they 
used (I, for example had to look up Rho, and there are several others like this for which I’m not sure 
the average reader will know what the authors mean with these).  

The authors state that ‘Overall, genome-wide differentiation between lyrata diploids and the tetraploid 
populations was extremely low (mean Rho between ploidies = 0.19)’. Low compared to what? It is the 
highest value in the table?! Table 1 needs the same statistics for diploid vs. tetraploid A. arenosa. I 
think this is what they compare it with?  

Fig. 2A should be better explained. What is the red line? There are two y-axes, however, which one is 

which (dots/line)?  

‘..tetraploids, Lwt hereafter: PIL, SCB, KAG, SWA, LOI, MAU). ‘ What happened to the GYE population? 
It is included in the STRUCTURE analysis but not in any of the scans. Or am I missing something?  

Figure 3 shows clear signal for introgression from lyrata to arenosa (topo 11) as well, which doesn’t 
overlap with genome scans and is probably not involved in WGD adaptation, but rather may be local 
adaptation. There is a room to explore that. The question is: is introgression adaptive only in the 
direction from arenosa to lyrata (for adaptation to WGD), or can it also be adaptive in the opposite 
direction (maybe local adaptation)?  

Reviewer #2 (Remarks to the Author):  

This manuscript studied the molecular basis of parallel adaptation to WGD in lyrata compared to 
arenosa. In order to address this question, authors used 92 genome sequences from 92 individuals of 
lyrata, arenosa and outgroups, and cytologically assessed meiotic stability and selective sweep 
analysis and looked the overlap between sweep and introgression. Overall, authors suggested that the 
molecular basis by which WGD was stabilised in lyrata and arenosa is shared, and the whole story is 
generally interesting.  
Comments and suggestions:  
1) I am not sure it is reasonable to say rescued the nascent A. lyrata tetraploids from early extinction, 
such as in abstract. At least, so far we do not have enough evidence to claim a newly originated 
tetraploids is easy to extinct because of meiotic stability. Authors might need to rephrase somehow.  
2) Besides meiotic stability related genes, from the selection sweep analysis, authors must have found 
other genes, it will be great to characterize those genes as well, which must be very important for the 
polyploidy's survive as well.  
3) Again, in the introgression section, besides meiotic stability related genes, what are the other 
genes? Which kinds of GO terms authors enriched? These are all important detailed information 
needed for the story  
4) 1% outliers from genome scan, this has to be explained more carefully, it would be great to 
perform some additional permutation to support this cut-off, or to explain this in detail somehow.  

Reviewer #3 (Remarks to the Author):  

The manuscript by Marburger et al titled “Interspecific introgression mediates adaptation to whole 
genome duplication” describes analyses of genome sequences from diploid and tetraploid A. lyrata and 
A. arenosa individuals. This study investigates patterns of divergence between ploidy levels and 
species, signatures of selection associated with ploidy increase, and signatures of introgression 
between the two species. It concludes that an overlapping set of genes are under selection in 
tetraploids of the two species. These genes are also enriched for signatures of introgression between 
the species.  

Although this paper tells a nice story, my concern is that it is an incremental contribution to our 
understanding of ploidy evolution in Arabidopsis. The authors re-do many of the same analyzes they 
previously did in A. arenosa on A. lyrate and, maybe not surprisingly, find similar patterns. Analyses of 
introgression between these species have been done and it is widely accepted that hybridization is 
very common in sympatric plants and often results in adaptive introgression so I’m not sure that this 
finding sufficiently novel. I also found the patterns of direction of gene flow a bit unclear. Below are 

more specific comments about the paper (although it is difficult to refer to locations without page 
numbers).  

I think there is some excessively strong language that is unnecessary and distracting for example in 
the intro and abstract: “escape from extinction following the trauma of WGD..”  
“evolutionary genomics underscores the ubiquity of WGD”  
“ WGD and hybridization are dramatic mutations”  

In the population structure analysis, is there an explanation for why the outgroup “CRO” groups with 
arenosa? With 4 clusters does this separate out?  

Given the variation in admixture and the strong geographic structure to these populations it would be 
helpful to have a map that labels these populations or at least shows how the populations are grouped 
(LET, LWT). It is implied that admixture proportion is related to geographic proximity between 
tetraploids but it would be nice to have this be tested and to visually see what the patterns look like. 
Looking at reference 22 (Hohmann and Koch) I believe the pattern of introgression will be similar to 
figure 1. Is there anything new about the patterns of introgression in this study compared to that 
study?  

The authors seem to imply that polyploidization itself allowed for introgression between the two 
species but it seems that the geographic range overlap is more likely responsible for determining the 
extent of geneflow between species. It doesn’t look like the diploids overlap in range and therefore 
they don’t hybridize. The tetraploids do overlap and the more they overlap the more they hybridize. Is 
there any evidence that the WGD actually directly enabled or allowed for introgression?  

It’s unclear to me where the estimates of gene flow (alleles/generation) in the text come from as the 
figure (S2) and table (S2) have different numbers. All the other numbers suggest asymmetric gene 
flow from lyrata into arenosa but the text reports symmetric.  

I am unclear on the DFOIL test that was performed. As I understand it, DFOIL works on a symmetric 
tree (in this case first split is between species and second splits are between populations in a species). 
Based on Figure S5, it looks like their trees are not symmetric and arenosa is not analysed as a 
monophyletic group. How was this analysis done? It’s also unclear to me why these particular 
populations were chosen and why it is even interesting what the direction of gene flow is. 
Furthermore, if the gene flow is ancestral to the tetraploid split, it seems to make sense to use a 
tetraploid and diploid lyrata sample to determine direction of introgression with arenosa. I’d like to see 
a more justified explanation for what the analysis is and why it was done and how it relates to the 
structure analysis and the fastsimcoal2 analysis. Why is there such a clear sign of directional geneflow 
in DFOIL but not with fastsimcoal2?  

The authors find that meiosis remains unstable in lyrata. This instability segregates within populations. 
Is there any connection between the instability and the degree of introgression? Or the loci that are 
implicated in selection? Given the remaining instability in both species it seems that stability is still 
evolving and therefore selected alleles could have arisen recently in either of the two species and be 
spreading. It is unclear to me how the authors are interpreting the instability and how it fits into their 
story of adaptation and introgression after WGD.  

For the selection outlier analysis, why were the top 1% of the lyrata outliers investigated but only the 
top 0.5% of the arenosa outliers look at? Is the pattern different if only the top 0.5% of the lyrata are 
considered? I would like to see more numbers in reporting of these outlier results. It is stated that 
“the majority of known loci” were also found in arenosa but what are the numbers? Are the selected 
loci in lyrata statistically enriched for meiosis/endopolyploidy/transcription genes as in arenosa and if 
so I’d like to see some statistics for this. When comparing the outliers from the Let and the Lwt 
analysis the authors state that there is considerable overlap. I’m not sure < 1/3 of the genes is 

considerable overlap. It would be more effective to just state the overlap. It is also stated that “genes 
encoded by these loci include many involved in genome maintenance and meiosis” but I would like to 
know what those numbers are as well.  

I would appreciate a clearer explanation of the Twisst results. It is stated that there are three 
introgression-indicative topologies (3, 11,14) but 3 is the dominant species topology and is not 
consistent with introgression so this needs to be clarified or corrected. It is unclear how this topology 
based inference controls for incomplete lineage sorting (which is likely very common in these new 
lineages). Their narrow windows of introgression are even more suggestive of ILS. Another test based 
on allele divergence as well as topology would likely be a better indication of which regions are 
introgression and which ILS. The summary of the Twisst results are confusing to me as well. The 
authors state that “several meiosis-related” loci are outliers in Twisst and list 4 then state that “only 
4” are not outliers for twist but divergence. These are the same number of loci but discussed with 
different adjectives. The next sentence states that “the vast majority of these loci were indicated as 
undergoing specific gene flow”. This statement is again inconsistent with the numbers above. I think 
these numbers need to be more clearly and fairly stated.  

The overall conclusion of the direction of geneflow is very unclear to me. It seems that the Fastsimcol2 
results show more lyrata to arenosa gene flow, the Dfoil shows symmetric or more arenosa to lyrata 
gene flow and the Twisst analysis shows marginally more lyrata to arenosa gene flow. Yet, the authors 
conclude that there is adaptive introgression of alleles from arenosa to lyrata. I think there is no 
evidence that this should be the conclusion. The story about adaptive alleles for stabilizing WGD is 
even more unclear because the authors argued that some of these alleles came from the diploid 
populations (see figure 2). Together these inconsistencies point out how tenuous it is to make 
functional conclusions based on population genetic sequence data alone.  

In summary, the authors find evidence of selection in the genome of A. lyrata. Some of these outlier 
loci appear to be involved in meiosis and could be implicated in stabilization after WGD. This is exactly 
what was found in A. arenosa with the same analyses. The authors find evidence of introgression 
between the tetraploids in the same way that previous studies have found introgression. The 
introgression is likely bi-directional but the tests are inconsistent. Some regions showing discordant 
gene-tree topologies also contain regions that are outliers for selection. The major inconsistencies in 
their data include the direction of gene flow as indicated by the different tests, the timing of geneflow 
relative to polyploidization and genome stabilization, and the function of the introgression in actually 
stabilizing meiosis after WGD.  

The  
Figures:  
Fig. 1. Please explain your abbreviations in part A. It would be helpful to have the ploidy level 
indicated in part B (for example with different shapes) to clearly see how species and ploidy divide 
along the PC’s. It would also be helpful to see what the populations are that are later called LET and 
LWT. In part C the description says the lines are proportional to parameters in Fig S2 but the 
migration estimates (M5 and M6) are different in S2 but look the same in the manuscript figure.  

Fig. 2. I would like to see a scale bar on part B. It is also impossible to read the top axis.  

Fif 3. I would like a clearer description of what the “weighting” axis is in part C. It should also be 
stated that the colors in part B and C correspond to the colors of the topologies in part A. I think it 
should be explained why the 4 of the top 5 topologies were not looked at (top 10, 15, 2, 1).