Sottas et al. BMC Ecol Evo (2021) 21:41 BMC Ecology and Evolution https://doi.org/10.1186/s12862-021-01773-1 RESEARCH ARTICLE Open Access Gut microbiota in two recently diverged passerine species: evaluating the efects of species identity, habitat use and geographic distance Camille Sottas1*, Lucie Schmiedová1, Jakub Kreisinger1, Tomáš Albrecht1,2, Jiří Reif3,4, Tomasz S. Osiejuk5 and Radka Reifová1 Abstract Background: It has been proposed that divergence in the gut microbiota composition between incipient species could contribute to their reproductive isolation. Nevertheless, empirical evidence for the role of gut microbiota in speciation is scarce. Moreover, it is still largely unknown to what extent closely related species in the early stages of speciation difer in their gut microbiota composition, especially in non-mammalian taxa, and which factors drive the divergence. Here we analysed the gut microbiota in two closely related passerine species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia). The ranges of these two species overlap in a secondary contact zone, where both species occasionally hybridize and where interspecifc competition has resulted in habitat use diferentiation. Results: We analysed the gut microbiota from the proximal, middle and distal part of the small intestine in both sympatric and allopatric populations of the two nightingale species using sequencing of bacterial 16S rRNA. We found small but signifcant diferences in the microbiota composition among the three gut sections. However, the gut microbiota composition in the two nightingale species did not difer signifcantly between either sympatric or allopatric populations. Most of the observed variation in the gut microbiota composition was explained by inter- individual diferences. Conclusions: To our knowledge, this is the frst attempt to assess the potential role of the gut microbiota in bird speciation. Our results suggest that neither habitat use, nor geographical distance, nor species identity have strong infuence on the nightingale gut microbiota composition. This suggests that changes in the gut microbiota composi- tion are unlikely to contribute to reproductive isolation in these passerine birds. Keywords: Gut microbiome, Reproductive isolation, Diet, Habitat use, Passerines, Luscinia Background referred to as the gut microbiota [1, 2]. It has been shown Vertebrates harbour taxonomically and function- that the composition of the gut microbiota can have pro- ally diverse microbial communities in their intestines, found efects on the host’s physiology and morphology, as well as behaviour [3–8]. Moreover, between-species divergence in the gut microbiota composition could *Correspondence: [email protected] 1 Department of Zoology, Faculty of Science, Charles University, Viničná 7, play a role in the establishment of reproductive isolation 128 44 Prague, Czech Republic between species and thus in generating species diversity Full list of author information is available at the end of the article [6, 9, 10]. Despite recent intensive research on variation © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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BMC Ecol Evo (2021) 21:41 Page 2 of 14 in the gut microbiota composition within and between and prevailing diet [20, 35], the gut microbiota may vertebrate species [1, 11–14] the factors that generate have diferentiated between the two nightingale species the gut microbial diversity are still not sufciently under- in sympatry. stood, especially in non-mammalian taxa. Additionally, Rarely-occurring interspecifc hybrids between the it is largelly unknown how often closely related species common nightingale and thrush nightingale are viable, difer in the gut microbiota composition and thus how but their relative ftness compared to the parental spe- widespread the efect of the gut microbiota in speciation. cies has not yet been evaluated thoroughly. Neverthe- Variation in the gut microbiota composition may arise less, it is known that following Haldane’s rule, F 1 hybrid due to multiple factors including diferences in the host’s females are sterile while F 1 hybrid males are fertile diet [15–18], habitat [19–21] or geographical range [36–38]. It has been also documented that backcross [22–24]. In addition, host genes involved in the manage- hybrids are rarely present in the sympatric population ment of the gut microbiota can play important roles in [39] and that gene fow can occur between the two spe- structuring gut microbial communities [25–27]. All these cies [29, 40]. factors as well as a stable and long-lasting transfer of the To elucidate the factors shaping the gut microbiota gut microbiota from parents to progeny may generate variation in nightingales, we analysed the gut micro- divergence in the gut microbiota composition between biota profles in sympatric and allopatric populations of species. However, the importance of specifc factors in both species using high-throughput sequencing of bac- shaping gut microbiota diversity seems to difer among terial 16S rRNA. Unlike most studies on vertebrate gut diferent vertebrate lineages [28]. microbiota based on the analyses of faecal samples as Between-species divergence in the gut microbiota a proxy for intestinal samples, we analysed the micro- composition can contribute to the origin of reproductive biota along the whole small intestine to obtain a more isolation by multiple ways. First, host-associated micro- complex view of the gut microbiota composition in the biota may be involved in assortative mating and thus the two nightingale species. First, we tested whether the gut establishment of pre-mating reproductive barriers [6, 9]. microbiota composition difers between the two species Furthermore, interactions between the host genome and and whether there are any bacteria exhibiting host spe- the microbiome, between diferent microbes of the same cies specifcity, which would suggest that the gut micro- metagenome, or between diferent host’s genes involved biota could potentially contribute to the reproductive in the management of microbial communities can be isolation between the two nightingale species. Second, disrupted in hybrids [13]. Tis can cause gut microbiota we compared the level of interspecifc diferences in the dysbiosis in hybrid individuals, which can reduce their gut microbiota composition in sympatry and in allopatry. ftness and contribute to postzygotic isolation [10, 12, A higher divergence in sympatry would imply a stronger 13]. efect of habitat use or diet, while a higher divergence in Here we studied the gut microbiota variation in allopatry would indicate a stronger efect of geographical two closely related passerine bird species, the com- region on the gut microbiota divergence [41, 42]. Simi- mon nightingale (Luscinia megarhynchos) and the lar levels of divergence in sympatry and allopatry would thrush nightingale (Luscinia luscinia). Te two species suggest that the divergence in host genes involved in the diverged approximately 1.8 Mya [29] and their breed- management of the gut microbiota and/or long-term ing areas currently overlap in a secondary contact zone transfer of the gut microbiota from parents to progeny spanning across Europe [30], where they occasionally may cause a divergence of the gut microbiota between hybridize. Both species are migratory and difer in their the two nightingale species. To our knowledge, this study wintering grounds in sub-Saharan Africa [31]. Tey is the frst to focus on the gut microbiota composition both preferentially occupy dense shrubby vegetation in a pair of closely related avian species with incomplete (often close to water bodies) and feed mostly on insects reproductive isolation, and to examine its variation in [31, 32]. In allopatric regions they inhabit the same sympatric and allopatric populations. Our fndings could habitats, while in the sympatric region their habitat have important implications for understanding the fac- use and diet have partially diferentiated, presumably tors afecting variation in the gut microbiota composition to reduce interspecifc competition [32–34]. Common in birds and the possible role of gut microbiota diver- nightingales in sympatry occur more frequently in dry gence in avian speciation. habitats and feed mostly on Coleoptera, whereas thrush nightingales in sympatry prefer wet habitats and feed Results more often on Diptera [32]. Given that the gut microbi- We sequenced metagenomic DNA extracted from three