UA Open Access Publishing Fund / We examined a secondary contact zone between two species of desert tortoise, Gopherus agassizii and G. morafkai. The taxa were isolated from a common ancestor during the formation of the Colorado River (4–8 mya) and are a classic example of allopatric speciation. However, an anomalous population of G. agassizii comes into secondary contact with G. morafkai east of the Colorado River in the Black Mountains of Arizona and provides an opportunity to examine reinforcement of species’ boundaries under natural conditions. We sampled 234 tortoises representing G. agassizii in California (n = 103), G. morafkai in Arizona (n = 78), and 53 individuals of undetermined assignment in the contact zone including and surrounding the Black Mountains. We genotyped individuals for 25 STR loci and determined maternal lineage using mtDNA sequence data. We performed multilocus genetic clustering analyses and used multiple statistical methods to detect levels of hybridization. We tested hypotheses about habitat use between G. agassizii and G. morafkai in the region where they co-occur using habitat suitability models. Gopherus agassizii and G. morafkai maintain independent taxonomic identities likely due to ecological niche partitioning, and the maintenance of the hybrid zone is best described by a geographical selection gradient model.

The tench Tinca tinca (Linnaeus, 1758) is a valued table fish native to Europe and Asia, but which is now widely distributed in many temperate freshwater regions of the world as the result of human-mediated translocations. Spatial genetic analysis applied to sequence data from four unlinked loci (introns of three nuclear genes and mitochondrial DNA) defined two groups of populations that were little structured geographically but were significantly differentiated from each other, and it identified locations of major genetic breaks, which were concordant across genes and were driven by distributions of two major phylogroups. This pattern most reasonably reflects isolation in two principal glacial refugia and subsequent range expansions, with the Eastern and Western phylogroups remaining largely allopatric throughout the tench range. However, this phylogeographic variation was also present in European cultured breeds and some populations at the western edge of the native range contained the Eastern phylogroup. Thus, natural processes have played an important role in structuring tench populations, but human-aided dispersal have also contributed significantly, with the admixed genetic composition of cultured breeds most likely contributing to the introgression. I have then designed novel PCR-RFLP assays...

La question abordée est celle de la structure génétique des anchois Atlanto-méditerranéens analysée grâce à 6 locus microsatellites et un gène du cytochrome b de l'ADN mitochondrial sur un échantillonnage dont quatre en provenance de lagunes réparti entre la Tunisie, la France, la Mer Noire et le Maroc. Deux locus microsatellites parmi les six étudiés révèlent une forte différenciation liée à l'habitat lagunaire. L'étude du cytochrome b de l'ADN mitochondrial montre la présence de deux clades mitochondriaux trouvés en sympatrie en fréquence variable dans tous les échantillons. L'analyse combinée montre un déséquilibre nucléo-cytoplasmique significatif, confirmant l'existence d'incompatibilités génétiques partielles entre les deux entités. Ces deux entités génétiques doivent de ce fait être considérées comme des unités de gestion distinctes. / We study the genetic structure of Mediterranean anchovy by analyzing six microsatellite loci and the cytochrome b gene of mitochondrial DNA on anchovy populations with four lagoons, which are from Tunisia, Morocco, France and Black Sea. Two strongly differentiated loci and nucleo-cytoplasmic disequilibrium revealed a differentiation related to a lagoon habitat. The study of cytochrome b mitochondrial DNA showed the presence of two mitochondrial clades found on sympatric with variable frequency in all samples. By contrast, four microsatellites were undifferentiated within the entire set of samples, testifying to a variable permeability of the two interacting genomes. Our findings confirm the existence of partial genetic incompatibilities between the two entities that should then be considered as distinct from a conservation point of view.

Speciation is an important process that is responsible for the generation of biodiversity on Earth. The importance of gene flow during speciation is hotly debated; however, it can be investigated by studying the process of intraspecific population differentiation. Here, I investigate the mechanisms that influence population differentiation and gene flow in brown (Sula leucogaster) and red-footed boobies (S. sula). These species are congeneric, broadly sympatric, and ecologically similar; however, they differ in their marine habitat preference (inshore vs. pelagic). I hypothesized that: (i) gene flow is restricted by major physical barriers in both species, and (ii) gene flow in the absence of physical barriers is more prevalent in red-footed boobies due to their pelagic marine habitat preference. First, I characterized the structure and function of the booby mitochondrial genome. Using extensive DNA sequencing and phylogenetic methods, I found that boobies have tandemly duplicated mitochondrial control regions that evolve in concert. I suggest that concerted evolution of duplicated control region may be common in seabirds. To quantify matrilineal population genetic structure and gene flow, I sequenced one control region in 513 brown and red-footed boobies. Both species exhibited strong population genetic structure that was attributable to continental barriers to gene flow such as the Isthmus of Panama. In the absence of physical barriers, gene flow was more prevalent in red-footed boobies. I also used microsatellite and nuclear intron loci to perform multilocus phylogeographic analyses of brown and red-footed boobies. Individuals of both species could be grouped into four genetic populations that corresponded to oceanographic regions. In red-footed boobies, gene flow following secondary contact of previously isolated lineages was common, perhaps due to a pelagic habitat preference. In brown boobies, major genetic populations apparently diverged in the absence of gene flow. Despite this, Isla San Benedicto in the Eastern Pacific is a site of secondary contact between brown booby lineages that diverged approximately one million years ago, and hybrids are apparently fertile. These findings suggest that gene flow following secondary contact may be an important, but overlooked, component of speciation. / Thesis (Ph.D, Biology) -- Queen's University, 2012-04-28 17:28:53.302

Les activités humaines sont à l’origine de profondes modifications de la distribution naturelle des espèces. Les introductions d’espèces sont en particulier à l’origine de contacts secondaires entre espèces non isolées reproductivement et ayant divergé en allopatrie. Cette situation est un cas d’étude particulièrement intéressant pour étudier des processus éco-évolutifs, tels que la coexistence d’espèces occupant de même niches écologiques ou les flux de gènes contemporains (i.e. hybridation et introgression) entre espèces indigènes et non-indigènes. Cette thèse s’est ainsi intéressée aux interactions écologiques et génomiques entre deux ascidies congénériques et interfertiles, Ciona robusta et Ciona intestinalis. Ces deux ascidies, abondantes dans les habitats portuaires, sont en sympatrie en Manche Occidentale suite à l’introduction récente de C. robusta (supposée originaire du Pacifique Nord-Ouest) dans l’aire de distribution naturelle de C. intestinalis. Par une étude de la distribution spatiale et temporelle (inter-saisonnière et inter- annuelle) des adultes des deux espèces et de la sédentarisation de leurs juvéniles, nous avons montré que les deux espèces coexistent de façon durable, au sein de communautés similaires, à l’échelle régionale. Elles vivent en syntopie dans la majorité des habitats portuaires étudiés, bien qu’il existe une importante dynamique saisonnière de leur abondance relative : C. robusta est surtout présente en fin d’été et en automne, et toujours en moindre abondance par rapport à sa congénère indigène. Cette syntopie, l’interfertilité des deux espèces et le synchronisme de leur maturité sexuelle indiquent un potentiel d’hybridation important entre les deux espèces. L’hybridation réalisée est pourtant faible, comme montré avec quatre marqueurs moléculaires diagnostiques des deux espèces, analysés sur plus de 3000 individus : seulement 4% des individus présentent des génotypes compatibles avec de l’hybridation ou de l’introgression. Ces résultats ont été complétés par une étude de génomique des populations (310 marqueurs SNPs et 450 individus collectés dans différents océans) : ils confirment que les flux de gènes interspécifiques sont très rares. Par ailleurs, les rares locus présentant du polymorphisme partagé montrent un taux d’introgression hétérogène, et l’introgression est également observée dans des localités où seule l’une des espèces existe (ex. côtes américaines et chiliennes). L’ensemble des résultats génétiques et génomiques indiquent que le flux de gènes interspécifique observé résulte d’introgressions anciennes (probablement au Pléistocène) et non contemporaines. Une dernière étude menée à l’échelle mondiale avec des marqueurs mitochondriaux a par ailleurs permis de montrer que les processus d’introduction de C. robusta diffèrent selon les régions d’introduction et pose la question du statut réellement non-indigène des deux espèces dans certaines régions. Ainsi, cette thèse a montré la (quasi)-absence d’hybridation actuelle entre C. robusta et C. intestinalis, en situation de syntopie. Elle ouvre des perspectives quant à l’étude des mécanismes d’isolement reproductif entre ces deux espèces. La question du devenir à long terme des deux espèces, qui par leur coexistence étroite sont en compétition, est posée. Cette compétition pourrait être modulée par leurs préférences environnementales et les changements climatiques en cours dans l’Atlantique Nord. Enfin, cette thèse illustre comment des processus contemporains et anciens interagissent pour façonner la distribution et l’évolution des espèces, la structure génétique de leurs populations et l’architecture de leur génome. / Human activities severely alter species ranges that have been built on evolutionary time scales, and biological introductions promote secondary contacts between non-reproductively isolated species that were in allopatry. Such a situation is a very interesting case-study to examine eco-evolutionary processes, for instance coexistence between species sharing the same ecological niche or inter-specific gene flow (i.e. hybridization and introgression) between native and non-native species. This PhD thesis studied ecological and genomic interactions between two biologically similar, interfertile and congeneric tunicates, namely Ciona robusta and Ciona intestinalis. They are both abundant in harbours and marinas. They are sympatric in the Western English Channel because of the recent introduction of C. robusta (putatively native to Asia) in the natural range of C. intestinalis. Based on spatial and temporal (seasons and years) adult abundance data and examination of recruitment patterns of the two species, we showed that the two species are sustainably coexisting at a regional scale in Brittany, within similar communities. They live in syntopy in most of the study marinas. However, there are significant changes in their relative abundance through time: C. robusta is mostly present at the end of the summer and in the autumn, although always being less abundant than its native congener. Syntopy, interfertility between the two species and synchronous sexual maturity all indicated a high likelihood of hybridization between the two species. The realized hybridization is however very low, as shown by 4 species-diagnostic molecular markers genotyped over more than 3000 individuals: only 4% of the individuals displayed a genotype compatible with hybridization or introgression. This result has been confirmed with a population genomics study (310 SNPs; 450 individuals collected worldwide): inter-specific gene flow is very low. In addition, the few loci showing shared polymorphism displayed variable introgression rates and introgression was observed even in allopatric localities (where only one of the two species exists nowadays). Overall, the genetic and genomic results indicate that interspecific gene flow is most likely due to past introgression events (that may have occurred during the Pleistocene). Finally, a study carried out at a worldwide scale with two mitochondrial loci, showed that the introduction processes of C. robusta are different among the introduced regions. This study also questions the non-native status of the two Ciona species in some regions. This work showed the near absence of contemporary hybridization between C. robusta and C. intestinalis, even in syntopy, opening new research perspectives about the mechanisms preventing their reproduction in the wild. It also questions the fate of these two competing species; this competition may depend on the interaction between their specific environmental preferences and on on-going climatic changes in the North Atlantic. Altogether, this work illustrates the interplay between contemporary and past processes on species distribution and evolution, population genetic structure and genomic architecture.

Despite more than 50 years of research on the genetic basis of speciation, we still know very little about the early stages of this process. A general rule of speciation is the Haldane’s Rule, which states that postzygotic isolation is expressed earlier in the heterogametic sex: sterility and inviability in hybrids is much more likely to occur in the heterogametic sex. However, it is still unknown how long hybrid sterility takes to be established in the heterogametic sex, since most studies have focused on highly divergent species that no longer hybridize in nature. The meadow grasshopper (Chorthippus parallelus) is a suitable system to shed light on this matter because hybrid male sterility exists between subspecies that show low divergence and are still able to hybridize readily in nature. C. p. parallelus is distributed throughout Northern, Central and Eastern Europe while C. p. erythropus is restricted to the Iberian Peninsula. Both subspecies meet in the Pyrenees, where they form a narrow hybrid zone. Yet, it is unknown 1) how long ago have these subspecies diverged and 2) how have they expanded to form the hybrid zone where these incompatibilities take place. Here, we applied phylogenomic methods to estimate the time of divergence of the subspecies and to study its phylogeographic history. Using the mitogenome and known mitochondrial rates of evolution, we dated the diversification of the subspecies ≈100,000 years ago and found six ancient mitochondrial haplotypes. Implementing coalescent theory to estimate a nuclear species tree, we found evidence of sub-refugia within two or three main refugia and that the hybrid zone originated from a recent post-glacial expansion from one sub-refugia. Haldane’s rule evolved rapidly in C. parallelus, within ≈100,000 years, with demographic processes, such as population expansion. Founder effect reduced genetic diversity in C. parallelus parallelus, with the possibility of fixing incompatible alleles with C. parallelus erythropus resulting in hybrid male sterility.

Les changements phénotypiques et génétiques consécutifs au processus de domestication et plus largement du passage d'un environnement sauvage aux agro-écosystèmes sont bien décrits chez les espèces végétales. Cependant, l’impact de ces changements de traits d'histoire de vie sur les populations de pathogènes infectant les hôtes domestiqués a jusqu’alors été très peu abordé. En particulier,quelles sont les conséquences sur les traits d’histoire de vie des pathogènes du passage d’un milieu sauvage caractérisé par une grande hétérogénéité d’hôtes à des agro-écosystèmes bien plus denses et homogènes génétiquement? Est-ce que le génome des pathogènes porte des signatures de changements démographiques ou de sélection en relation avec la domestication de leurs hôtes ? Ces questions traitées dans le cadre de la présente thèse ont porté sur le champignon Venturiainaequalis, agent pathogène de la tavelure du pommier, qui partage en Asie Centrale le même centre d’origine que son hôte endémique sauvage Malus sieversii. Les travaux reposent principalement sur 1)l'étude du passage de souches sauvages virulentes sur pommier résistant VF expliquant le contournement rapide de cette résistance et 2)l'étude de génomique des populations et de variations phénotypiques comparant une population de V. inaequalis échantillonnée sur M.sieversii au Kazakhstan, préalablement identifiée comme étant une relique de la population ancestrale, et d'une population Kazakhe de milieu anthropisé. Ces deux populations sont génétiquement différenciées et partiellement isolées reproductivement. L’utilisation des deux méthodes d’inférence l'une l'ABC et l'autre dadi basées sur l’alignement de 10 994 gènes échantillonnés au sein des deux populations révèlent une histoire complexe de contact secondaire, où le champignon aurait dans un premier temps « suivi » la plante hôte à travers le monde au cours de la domestication (modèle de l'hosttracking) puis échangerait à nouveau depuis peu des gènes avec la population ancestrale au Kazakhstan. Cette situation originale de remise en contact secondaire favorisant la mise en évidence d’incompatibilités génétiques, permet à la fois d’envisager de détecter des gènes impliqués dans l’adaptation à l’habitat (pommier cultivé versus Malus sieversii) et dans l’isolement reproductif post-zygotique. L’analyse des 36 génomes séquencés a ainsi permis d’identifier plus de 602 gènes présentant un indice de différentiation (Fst) supérieur à 0,7 autant de gènes verrous faisant potentiellement obstacle aux flux de gènes homogènes entre ces deux populations. Enfin, l’analyse phénotypique de ces deux populations montre que la domestication du pommier a profondément modifié les traits d’histoire de vie de V. inaequalis liés à sa dispersion. L’ensemble de ces analyses a donc permis d’identifier des locus candidats potentiellement impliqués dans des modifications des traits d’histoire de vie, et dans des barrières géniques, en lien avec la domestication du pommier. / Phenotypic and genetic changes occurring during the process of domestication are well described in plants. However, the impact of domestication in life history traits of their pathogens has been poorly studied. In particular, what are the consequences on the life history traits of pathogens that switch from the wild habitat characterized by a high host genetic and spatial heterogeneity to a much more dense and genetically homogeneous agroecosystems? Have pathogen’s genomes particular signatures of demographic changes or of selection related to the domestication of their host? Here we focused on the fungus Venturia inaequalis, causal agent of apple scab, that shares in Central Asia the same center of origin of its wild endemic host Malus sieversii. In the first part of this work we retrace evolutionary history of a population from the wild that was responsible for the rapid breakdown of the Vf resistance gene in apple orchards. We then highlight the threat of wild habitat to scab resistance apple cultivars and thus the necessity to take into account the wild in the management of resistance genes. In the second part, a comparative study based on phenotypic and genomic data was carried out between two populations sampled in Kazakhstan on M. sieversii, either in anthropized areas or in non anthropized area, the latter being identified as a relic of the ancestral population of V. inaequalis. These two populations were genetically differentiated and partially reproductively isolated. The use of two methods of inference (ABC and dadi) based on the alignment of 10 994 genes sampled in the two populations revealed a complex history of a secondary contact event. The fungus would have first followed its host worldwide by "Host-tracking" during the domestication process and then very recently it would re-exchange genes with its ancestral population in Kazakhstan. Secondary contact context is particularly favorable to detect genetic incompatibilities, this particular situation could then allow identification of genes involved in adaptation to habitat (cultivated apple versus M. sieversii) and in post-zygotic reproductive isolation. Analysis of 36 sequenced genomes has identified more than 602 genes with an index of differentiation (Fst) greater than 0.7, what represents numerous candidates genes of potential barriers to homogeneous gene flow between these two populations. Comparative phenotypic analysis between these two populations such spores size and capacity to sporulate showed that apple domestication would have also modified life history traits of V. inaequalis related to its dispersion. This study has allowed identification of candidate loci potentially involved in changes in life history traits, and genetic barriers in pathogen related to the domestication of apple.

In this collection of papers I have summarized my investigations into the field of evolutionary genetics and more specifically into patterns of biodiversity and evolutionary processes. The lizards (and frogs) studied here share common features in that they are largely present in unique environments, which are also regions that are biologically understudied. Most of these taxa show high degrees of endemism, interesting natural history characteristics, and each group manifests distinctive adaptations of general evolutionary interest. My work in the genus Telmatobius has been a progressive approach that began in my MS program, and it first focused on alpha taxonomy, morphological variation, and species boundaries. This work led to new studies initiated and completed at BYU involving further taxonomic revision (Formas et al., 2003; Chapter 1), and then revisiting and re-evaluating species boundaries established earlier (with allozyme markers) and this time with population level molecular (mitochondrial DNA) markers (Chapter 2). Our results indicate that the striking differences in size, coloration and general appearance in the various Lake Titicaca morphotypes are not genetically based. Further, there is evidence that these morphotypes have evolved very rapidly after demographic bottlenecks eroded present genetic variability. Telmatobius frogs of Lake Titicaca are listed by the International (IUCN) as critically endangered. We support this classification and further suggest studies to explore open questions like the possibility of adaptation along ecological resource gradients. Lizards of the genus Microlophus are interesting but for different reasons, and studies of this group constitutes the bulk of my dissertation work. The genus includes both Galapagos insular species, and continental taxa distributed in a linear gradient along > 4000 km of the western coast of South America. In studying Microlophus I first tackled the unresolved phylogenetic relationships within the genus (Chapter 3) and then pay attention to phylogeographic aspects of the most speciose lizard radiation in the Galapagos Archipelago (Chapter 4). Chapter 3 is a single manuscript provisionally accepted in the journal Systematic Biology. This paper introduces the lizard genus Microlophus (“lava lizards”) as a study system, and includes a large nuclear data set accompanied by an equally large mitochondrial data set (7877 characters in total). This paper explicitly differentiates among sequence alignments of gene regions that vary in tempo and class of mutational events. We show that this recognition is important and we suggest ways to appropriately deal with the alignment of multi-locus non-coding DNA data sets. A secondary finding in this study is that mtDNA and nDNA topologies are discordant with each other but that both are strongly supported, and that the nuclear topology is concordant with species distribution patterns along coastal South America. We hypothesize that in this particular region of the tree, the nuclear genome recovers a topology that is closer to the species tree, and conflicts occur due to likely secondary contact of distantly related taxa, suggesting that unique taxonomic relationships in the mtDNA gene tree are the result of hybridization. This last point highlights the value of dense taxonomic and character sampling for teasing apart different aspects of evolutionary processes. Chapter 4 is a manuscript to be submitted to the journal Evolution; in this study we further investigate the most speciose radiation of Microlophus in the Galapagos, based on an unparalleled sampling of most islands and small islets in the Archipelago. We use mtDNA sequences to both test hypothesized between-island colonization routes, as well as the expectation that within-island phylogeographic structure should be greater on older islands. Our mtDNA gene tree is strongly supported and allows rejection of previous alternatives, and we propose a novel sequence of between-island colonization events. Our results also reject the idea of phylogeographic structure been related solely to island age. Instead, we provide evidence to suggest that active volcanism as a major player in the generation of genetic diversity in within-island environments, and this is further compounded by the seemingly stochastic nature of within-island long-distance colonization routes mediated by ocean currents. We suggest that the direction and intensity of these currents, as currently understood, are insufficient to generate a priori hypotheses of oceanic colonization routes and their influence on gene flow. We do show that the standard stepping-stone model of migration, where genetic interchange is only possible among neighboring localities, does not explain much of the within-island population genetic structure unraveled by this study. From a biological conservation perspective the study of patterns of recent evolutionary history in the Galapagos provides with a window to evolutionary processes that have shaped and continue to impact the generation of biodiversity in the Galapagos Archipelago. Islands have long been viewed as natural laboratories of evolutionary change, and thus all island isolates are or could be distinctly important components of the larger, archipelago-wide processes. We provide working hypotheses for some of the demographic processes that might be generating within- and between-island biodiversity in this clade of lizards; confirmation of these explanations with independent data will have management implications for conserving the unique patterns observed in the Galapagos biota, but also the processes that generated these patterns.

A critical mechanism underpinning current biological diversity is the extent to which one species mates with, or avoids mating with, another. However, little is known about the factors that mediate hybridisation, especially during the initial and rarely observed stages of secondary contact when interspecific interactions have not responded to selection. In particular, whilst hybridisation is ultimately a behavioural phenomenon, the role of behaviour in mediating hybridisation and how it is influenced by environmental and circumstantial factors is rarely investigated. Recently introduced species provide us with unequalled opportunities to study these factors. In this thesis I examine the role of behavioural mechanisms, in particular male-male competition and mate choice, in mediating mating patterns between two genetically and phenotypically distinct lineages of European wall lizard (Podarcis muralis) that have come into recent secondary contact through human introductions. In Chapter Two, I investigated how sexual selection during allopatry is responsible for creating stark differences in phenotypic traits such as body size and weapon performance evident in the two lineages today, ultimately explaining the strong biases in dominance during territorial disputes between males. However, I also show that even given this asymmetry in male competitive ability, the extent to which it extrapolates into greater access to females in naturalistic, outdoor enclosures depends strongly on the spatial clustering of basking sites, a critically important resource for many ectotherms. In contrast to initial predictions suggested by asymmetries in male competition outlined in the previous chapter, in Chapter Three I show that both paternity and courtship behaviour was strongly assortative in the outdoor enclosures. Further investigation through staged experiments on olfactory mate choice, mating trials and analyses on specific behavioural data obtained in an enclosure experiment, I show that lineage based dominance actually contributes to assortative mating patterns in conjunction with weak conspecific male choice. In contrast, female choice seems to play no role in mediating the mating patterns observed between the two lineages. In Chapter Four I had the rare opportunity to examine the morphological and behavioural factors that predict why animals should hybridise in the first place, using the data obtained in the enclosure experiment above. I found that hybridisation was particularly common between small individuals of the larger lineage and large individuals of the smaller lineage; a result that corroborates the mechanisms determining the assortative patterns uncovered in Chapter Three. Additionally, hybridisation rates were particularly high in less dominant individuals, which I suggest is due to subordinate males having reduced opportunities for courting conspecific females due to male-male competition, requiring them to become less ‘choosy’ and therefore more likely to mate with heterospecifics. Finally, secondary contact cannot occur without at least one lineage coming into a new environment, and yet relatively little attention is paid to how this environmental change can affect the signals involved in intraspecific communication and mate choice. In Chapter Five I show that a change in the amount of time male lizards spend thermoregulating (a likely consequence of arriving in a new environment) significantly changes the chemical composition of their scent marks. However, whilst female lizards were able to detect these effects, they did not seem to base their mating decisions on them. Nevertheless, this result raises interesting questions about the potential function and consequences of this plasticity, and highlights the importance of considering plasticity in chemical communication in heterogeneous environments. Overall, this thesis shows the critically important role of behaviour in mediating intra- and interspecific mating patterns during recent secondary contact. In particular, it highlights how the direction and extent of hybridisation and competition are influenced by the degree to which differing morphological and behavioural phenotypes interact over a heterogeneous environment, particularly during the initial stage of secondary contact when mate choice has not had the chance to respond to the selective pressures of hybridisation.

Climate change is affecting the distribution of species, sometimes making previously separated species come into secondary contact. For closely related species, this often leads to competition and sometimes also hybridization, which can have negative effects on genetic bio? diversity. Collared flycatcher (Ficedula albicollis) and pied flycatcher (Ficedula hypoleuca) are two closely related species that hybridize where they meet on the Swedish islands Gotland and Öland. Studies from Gotland and the north of Öland have shown that the pied flycatcher is outcompeted and displaced from favourable, high-quality habitat by the collared flycatcher, leading to a decrease in the number of pied flycatchers in deciduous forest. However, this has not been shown for the ecologically important and one of the largest deciduous forests in Sweden, the Mittland forest located in central Öland. Here I use census data collected during two periods separated by 37 years from the Mittland forest to investigate habitat choice and competition between the two species. Although the number of collared flycatcher has greatly increased in the area, I show that the collared flycatcher has not outcompeted the pied flycatcher from favourable habitat. I also show that pied flycatcher does not have a preference for coniferous forest. Lastly, I show that the two species have a near complete overlap of habitat niches. These results indicate that they are in direct competition over the favourable habitat, that there is no equilibrium between the species and the collared flycatcher might still be expanding in the Mittland forest. Allowing more time to pass and further research will show if the pied flycatcher will be displaced from the high-quality habitats in this area or not.