The evolution of transposable elements in Brachypodium distachyon is governed by purifying selection, while neutral and adaptive processes play a minor role

Reviewer #1 (Public Review):

The study presented in this manuscript presents very convincing evidence that purifying selection is the main force shaping the landscape of TE polymorphisms in B. distachyon, with only a few putatively adaptive variants detected, even though most conclusions are based on the 10% of polymorphisms contributed by retrotransposons. That first conclusion is not novel, however, as it had already been clearly established in natural A. thaliana strains (Baduel et al. Genome Biol 2021) and in experimental D. simulans lines (Langmüller et al. NAR 2023), two studies that the authors do not mention, or improperly mention. In contrast to the conclusions reached in A. thaliana, however, Horvath et al. report here a seemingly deleterious effect of TE insertions even very far away from genes (>5kb), a striking observation for a genome of relatively similar size. If confirmed, as a caveat of this study is the lack of benchmarking of the TE polymorphisms calls by a pipeline known for a high rate of false positives (see detailed Private Recommendations #1), this set of observations would make an important addition to the knowledge of TE dynamics in the wild and questioning our understanding of the main molecular mechanisms through which TEs can impact fitness.

Reviewer #2 (Public Review):

Summary:
Transposable elements are known to have a strong potential to generate diversity and impact gene regulation, and they are thought to play an important role in plant adaptation to changing environments. Nevertheless, very few studies have performed genome-wide analyses to understand the global effect of selection on TEs in natural populations. Horvath et al. used available whole-genome re-sequencing data from a representative panel of B. distachyon accessions to detect TE insertion polymorphisms (TIPs) and estimate their time of origin. Using a thorough combination of population genomics approaches, the authors demonstrate that only a small amount of the TE polymorphisms are targeted by positive selection or potentially involved in adaptation. By comparing the age-adjusted population frequencies of TE polymorphisms and neutral SNPs, the authors found that retrotransposons are affected by purifying selection independently of their distance to genes. Finally, using forward simulations they were able to quantify the strength of selection acting on TE polymorphisms, finding that retrotransposons are mainly under moderate purifying selection, with only a minority of the insertions evolving neutrally.

Strengths:
Horvath et al., use a convincing set of strategies, and their conclusions are well supported by the data. I think that incorporating polymorphism's age into the analysis of purifying selection is an interesting way to reduce the possible bias introduced by the fact that SNPs and TEs polymorphisms do not occur at the same pace. The fact that TE polymorphisms far from genes are also under purifying selection is an interesting result that reinforces the idea that the trans-regulatory effect of TE insertions might not be a rare phenomenon, a matter that may be demonstrated in future studies.

Weaknesses:
TEs from different classes and orders strongly differ in multiple features such as size, the potential impact of close genes upon insertion, insertion/elimination ratio (ie, MITE/TIR excision, solo-LTR formation), or insertion preference. Given such diversity, it is expected that their survival rates on the genome and the strength of selection acting on them could be different. The authors differentiate DNA transposons and retrotransposons in some of the analyses, the specificities of the most abundant plant TE types (ie, LTR/Gypsy, LTR/Copia, MITE DNA transposons) are not considered.

The authors used a short-read-based approach to detect TIPs and TAPs. It is known that detecting TE polymorphisms is challenging and can lead to false negatives, depending on the method used and the sequencing coverage. The methodology used here (TEPID) has been previously applied to other species, but it is unclear if the sensitivity of the TIP/TAP caller is equivalent to that of the SNP caller and how these potential differences may affect the results.