Reviewer #1: Dr. Scott Roy (nominated by Dr. W. Ford Doolittle)
Reviewer’s comments: The authors provide a very useful and timely review on the mechanisms of intron gain, a field in which a tremendous amount of progress has been made in the past few years. I have very few criticisms, and even on those points where I might have a difference of opinion in regards to emphasis or interpretation, I think the authors' perspective is well supported and balanced, and so overall I do not think any changes need to be made.
Authors’ response: We thank you for your review and comments. Because most of these mechanisms lack definitive evidence to support how/if they occur, we agree that there exist various, and at times conflicting, opinions and interpretations upon how/if these proposed mechanisms occur. Thus, we are delighted that you feel our presentation was balanced and supported by primary literature.
Reviewer #2: Dr. Eugene Koonin
Reviewer’s comments: This is a timely review of the important and fascinating problem of intron gain routes and mechanisms. The article is very well structured around the 7 distinct (proposed) routes of intron gain. My mild disappointments have to do with the excessive brevity of some of the sections. In particular, the section on Group II intron insertion is succinct to the point of being potentially misleading. I agree with the authors that there is no indication of recent intron gain via Group II intron insertion. However, this does not put into doubt the ultimate origin of spliceosomal introns from Group II introns whereas from the current version, the impression is that the authors refute that scenario. The situation can be easily remedied with a brief recapitulation of the evidence in support of the evolutionary links between self-splicing and spliceosomal introns.
Authors’ response: We intentionally kept our review brief in order to meet the word limit of reviews in this journal (~3000 words). We apologize if, in our desire to write succinctly, we may have accidently expressed our beliefs and prior findings incorrectly, especially in regards to the origin of spliceosomal introns. As you mentioned, a significant amount of evidence exists to support the theory that group II introns, originating from an α-proteobacterium that would later go onto serve as the mitochondria, invaded the genome of their archaeal host, and in response to this invasion, selective pressures eventually gave rise to the nucleus, nonsense-mediated decay, and spliceosomal introns. We do note that in our original manuscript we directly stated that “group II introns are widely regarded as the progenitors of spliceosomal introns”. However, the wording of this section may have been misleading. Thus, we have rewritten the section “Insertion of Group II Intron” to better exemplify this point and have also added a brief primer on the origin of spliceosomal introns.
Reviewer’s comments: In addition, the authors do not explicitly address the connection between alternative splicing and intron gain mechanisms. Emergence of a new alternative splice form often involves ("part-time") intronization, so I think this belongs in the paper.
Authors’ response: Alternative splicing and, in particular, nonsense-mediated decay likely play key roles in the evolution of gained introns by alleviating negative fitness costs imposed upon organisms following the initial intron gain event (or, more explicitly stated, the initial mutation). However, due to space limitations, we chose to focus only on the mechanisms that may initially cause the intron gain (the initial mutation). The forces at play following this mutation that fixate this mutation in a population/species deserve a more in-depth treatment than we could provide with the space allotted. Regardless, we have added a brief discussion of these topics to the “Intronization” section, as this mechanism may rely most heavily upon evolutionary processes to yield efficient introns.
Reviewer’s comments: Again, a very timely and useful review but with a little more attention to details, it could become even better.
Authors’ response: We thank you for your review and apologize if we may have neglected some topics in an effort to be succinct.
Reviewer #3: Dr. John Logsdon, University of Iowa
This reviewer provided no comments for publication.