A review of Ph.D. thesis entitled: The role of interspecific competition in ecological differentiation and speciation in two passerine species, Luscinia megarhynchos and Luscinia luscinia, by Camille Sottas. This thesis focuses on the biology of two recently diverged species – the common nightingale and the thrush nightingale. The student took advantage of joining a strong scientific team with previous experience, data and continuous funding of the project. The obtained results are interpreted in the context of interspecific competition, character displacement and speciation. The thesis consists of three scientific papers and the student is the first author of all of them. Two papers have already been published in respected scientific journals and the quality of the third contribution is also high so I expect its soon publication as well. First chapter deals with interspecific differences in habitat use and bill size in allotopic and syntopic sites within a zone of sympatry. Habitat of both species was similar on syntopic sites whereas different on allotopic ones which pattern was mirrored in the bill size. Second chapter expands these ideas and investigates whether the variation in bill size can be explained by the diet. The diet was indeed correlated with bill morphology. However, this pattern was largely driven by species identity. Thrush nightingale consumed more flies and had shorter bill than common nightingale who consumed more beetles and had longer bill. The third chapter deals with gut microbiota which could depend on the diet and have consequences for the speciation process. However, the differences between species were smaller than those between individuals of the same species suggesting that gut microbiota is individual- rather than species-specific trait. I like the integrative nature of this thesis very much. Diverse approaches were employed to answer research questions. Morphology of both species and habitat characteristics of their territories were investigated in the field. Prey was identified to species and measured under the dissecting scope in the laboratory. This task was surely time-demanding as the prey items were numerous and fragmented. These classical methods were combined with the modern molecular tools including prey identification by barcoding and sequencing of 16s ribosomal DNA to describe gut microbiome. The data were appropriately analyzed. All papers are also very well written with a strong story making their introductions and discussions attractive to the general readership. The same holds for the introductory part of the thesis that was solely written by the student who thus proved her competence of scientific writing, including very high standard of English language. Taken together, this dissertation certainly fulfills all requirements for Ph.D. theses and I recommend it to be the subject of the defense process. Comments and questions: Comment 1: In my view, the strong story of all contributions has not always been supported by comparatively strong evidence. Although the whole thesis and its first two chapters included the term „interspecific competition“ in their titles, I found only limited evidence for this process in the thesis at best. The thesis does not contain any behavioral data that could directly prove interspecific interference competition between these species (cf. Chapter I, p. 918). The operation of interspecific competition is also suggested when species diverge more in sympatry compared to allopatry. This point was clearly articulated in the introduction of the first chapter. After reading this, I was surprised that this section ends with a prediction: „...the species will differ in both habitat use in allotopy, but not in syntopy“ (Chapter I, p. 915). This prediction does not follow from the introduction, it seems to be even in contrast to it, although it is in line with the results. However, I think that interspecific competition is not necessary to explain results of this study. The parsimony explanation may be that these species evolved different habitat preferences when in isolation. Subsequent expansion of their ranges brought these species into secondary contact only at sites that represent intersection of their habitat preferences. Consequently, habitat on syntopic sites is expected to be more similar than that on allotopic sites. Furthermore, similarity of habitats of the two species on syntopic sites is also expected by the definition of syntopy and spatial autocorrelation. Anyway, I suppose that habitat differences between syntopic and allotopic sites are only indicative of habitat segregation between the species and tell nothing about interspecific competition. Comment 2: Similarly, I see little evidence that interspecific competition shaped the differences in the diet and bill morphology between the two species (chapter II). The only tentative support for the role of interspecific competition came from a re-analysis of older data that suggested greater divergence of bill size in sympatry than in allopatry (appendix S2). Authors found significant interaction between region (sympatry vs. allopatry) and species on bill size. However, I have doubts whether this interaction is the relevant test for „increased divergence in sympatry compared to allopatry...“ (main text of chapter II). The significance of this interaction is likely driven by opposite effects in sympatry where thrush nightingales have longer bills and allopatry, where common nightingales have longer bills. As far as I know, the concept of character displacement deals with an absolute difference between characters, not a signed one. In other words, the character displacement does not predict different direction of the difference in sympatric and allopatric populations. Comment 3: In chapter III (page 13) authors misinterpret their own results when write: „Our previous research documented that sympatric populations of common and thrush nightingales in the zone of their secondary contact exhibited higher divergence in habitat use (Reif et al. 2018; Sottas et al. 2018, 2020) and bill morphology (Reifová et al. 2011) compared to allopatric population“. However, both Sottas et al. 2018 and 2020 were done exclusively in sympatry. Moreover, habitat was more similar on syntopic compared to allotopic sites (see above). Comment 4: Camille Sottas declares that she contributed to the fieldwork in all three studies included in this thesis. However, the first chapter (Sottas et al. 2018) is based on data gathered in the field in 2007, i.e. before the start of this PhD study and thus probably without the help of this student. Comment 5: Alatalo et al. (1990, Anim. Behav, 39, 601-603) did not study interspecific competition between collared and pied flycatcher but only mate attraction in the latter species (cf. Introduction, p. 5). As Alatalo et al. study has been misinterpreted in the same way in another study cited by the student (Rybinski et al. 2016) it seems that the former source was borrowed from the latter one without reading. Question 1: One interesting result is the dependence of bill size on habitat characteristics such as elevation (Chapter I). I wonder how these differences in bill length (heritable character) may be maintained on a small spatial scale despite potential gene flow between sub-populations? Similarly, authors argue that the differences in bill size of the two species are not driven by ambient temperature as this is not very variable within their study area (Chapter II). However, if the populations were panmictic, the whole breeding range would be important for bill evolution and ambient temperature surely differs between species on this global scale. Question 2: Chapter II: Nightingale diet was determined from faecal samples and regurgitates of adult males. As the competition for food may be most intense when nestlings are being fed, I wonder whether the food delivered to nestlings is expected to be the same as that consumed by adults. If not, can this have any consequences for thoughts about interspecific competition? Furthermore, these migratory species spend most of the year outside their breeding sites. Can the differences in their bill size, for example, be evolved due to habitat/diet differences on their wintering sites? These sites lay thousands of kilometers from one another and therefore may differ more than partly overlapping breeding sites of these species. Question 3: Authors suggested that „...the two nightingale species....escape from interspecific competition by moving to habitats avoided by the competitor“ (Chapter I, p. 919). I wonder if avoidance of the habitat where interspecific competitor is present can lead to breeding in habitats with higher density of and therefore competition from conspecifics. Intraspecific competition is usually more intense than interspecific one since individuals of the same species compete for everything, including mates. By this logic, individuals might avoid sites with a high density of conspecifics even if it would mean that they ended near interspecific competitors. Can you comment on this? Question 4: Nightingales used for analyses of gut microbiota were killed. I understand that this enabled to analyze gut microbiota in three gut sections which is advance over analyses made from non-invasive faecal samples. However, I consider this advance insufficient to justify killing of these songbirds. Were the killed birds used for any further objectives which would add to justification of their deaths? 3rd September 2020 Miloš Krist Department of Zoology and Laboratory of Ornithology Faculty of Science, Palacky University Olomouc .