Speciation is a fundamental concept in evolutionary biology, and understanding the mechanisms driving speciation remains the foremost research topic within this field. Hybridisation is often involved in speciation and can influence its rates, potentially accelerating, decelerating or even reversing the process. This study investigates the evolutionary history of the New Guinean bird genus Melidectes, consisting of six species that inhabit various montane regions at different elevations. While most Melidectes species have allopatric distributions, two species overlap in the central mountain range and hybridise. However, plumage differences and elevational adaptations are assumed to maintain the species' boundaries. Utilising specimens from natural history collections and comprehensive genomic analyses, including a de novo genome assembly, we characterise allopatric speciation patterns within the genus and highlight how future speciation could potentially be driven by climate change. Contrary to previous hypotheses, our findings suggest that in the two distributionally overlapping species, phenotypic differences do not prevent gene flow. We find limited acoustic differentiation and extensive admixture across most of their distributions. Divergence and admixture levels conform poorly to the current taxonomy and follow a geographical pattern in which the most isolated populations at the ends of the distributions are most divergent and show least admixture. However, in contrast, their mitochondrial genomes do group in accordance with species identity, namely, into two deeply divergent lineages. We propose that this system demonstrates the ephemeral nature of speciation, in which two incipient species have started mixing extensively as they came into secondary contact, resulting in nearly complete fusion into a single lineage.

Hybridisation is known to occur between a wide range of taxa, including species for which strong sexual selection has led to markedly different sexual phenotypes and lek-mating behaviours. To what extent occasional hybridisation can overcome the reproductive barriers in such systems and, for example, lead to the establishment of hybrid zones is poorly known. In this study, we address this question by focusing on one of the most well-known avian radiations in which sexual selection has resulted in an extraordinary assemblage of phenotypic diversity and lek-mating behaviours: the birds-of-paradise (Paradisaeidae). We quantify the genome-wide distribution of introgression and find multiple signals of recent and historical gene flow between and within two genera of birds-of-paradise, Astrapia and Paradigalla. In addition, we present the first empirical genomic indication of a putative hybrid zone between two lekking bird-of-paradise species that differ substantially in their sexually selected traits and behaviours. Our findings are consistent with the idea that behavioural and phenotypic traits may constitute weaker pre- and post-zygotic barriers to gene flow than generally thought in lek-mating species.

Genomic studies have revealed introgressive hybridisation as a common phenomenon across the tree of life, particularly among young radiations. As incipient speciation tends to be induced by vicariance events, it is assumed that introgressive hybridisation is more frequent in young radiations in which allopatrically distributed species have a high probability of coming into secondary contact. In this study, we use whole genomic data to investigate spatio-temporal introgression patterns in a songbird radiation that has colonised a highly dynamic island region in the Indo-Pacific. Some taxa within this radiation have colonised remote oceanic islands whereas others occur on landmasses and islands in the Sahul region that were periodically connected during Pleistocene periods of lower sea levels. Our results show that introgressive hybridisation has been pervasive within this young radiation, despite prominent plumage differences between taxa. Geographical proximity has been an important factor for hybridisation and we further find that species occupying islands in the environmentally unstable Sahul region exhibit particularly high signatures of introgressive hybridisation. Yet, one species appears to have been shielded from hybridisation, perhaps due to specific ecological specialisations. Finally, we identify a hybrid species on an island where two oceanic radiations meet. Our results also caution against relying solely on analyses that only detect asymmetric introgression when examining systems with complex introgression histories. Collectively, our results support a growing body of literature that suggests that reticulate speciation is more common than previously thought. This has implications for our understanding of species formation and their persistence through time.

Despite large differences in morphology, behavior and lek-mating strategies the birds-of-paradise are known to hybridize occasionally, even across different genera. Many of these bird-of-paradise hybrids were originally described as distinct species based on large morphological differences when compared to recognized species. Nowadays, these specimens are generally recognized as hybrids based on morphological assessments. Having fascinated naturalists for centuries, hybrid specimens of birds-of-paradise have been collected and the specimens kept in Natural History Collections. In the present study, we utilize this remarkable resource in a museomics framework and evaluate the genomic composition of most described intergeneric hybrids and some intrageneric hybrids. We show that the majority of investigated specimens are first-generation hybrids and that the parental species, in most cases, are in line with prior morphological assessments. We also identify two specimens that are the result of introgressive hybridization between different genera. Additionally, two specimens exhibit hybrid morphologies but have no identifiable signals of hybridization, which may indicate that minor levels of introgression can have large morphological effects. Our findings provide direct evidence of contemporary introgressive hybridization taking place between genera of birds-of-paradise in nature, despite markedly different morphologies and lek-mating behaviors.

New Guinea is home to one of the world’s most extraordinary families of birds; the birds-of-paradise (Aves: Paradisaeidae). The family consists of 45 recognised species and many of them have developed remarkable behavioural and plumage traits. Furthermore, the majority of the species reproduce through lek-mating, an extreme form of sexual selection in which female choice determines male mating success. While strong sexual selection is generally considered as a form of pre-zygotic isolation, interestingly, hybrids between distant lineages of birds-of-paradise are well documented across the family. Having fascinated naturalists for centuries, bird-of-paradise specimens have been collected and preserved in natural history collections, including different combinations of putative hybrid specimens. As such, natural history collections represent a remarkable resource for the study of the evolutionary and genomic consequences of interspecific hybridisation in birds-of-paradise. This thesis draws on specimens from natural history collections to study the prevalence of hybridisation and its effect on birds-of-paradise. In chapter I, we present a workflow designed for large scale use of avian museum specimens in genomic studies. The workflow was used to generate genome-wide re-sequencing data for the three other chapters of the thesis. In chapter II, we analyse 37 bird-of-paradise hybrids, originally determined to be hybrids based on morphology. We use genomic methods to verify the hybrid status and the parental species of each hybrid and show that the majority of the specimens are first-generation (F1) hybrids. However, we also identify two specimens that are the result of backcrossing and introgressive hybridisation between different genera. Our findings are the first direct evidence that contemporary introgressive hybridisation takes place between genera of birds-of-paradise in the wild and that interspecific hybrids are not necessarily sterile. In chapter III, we compile a population genomic data set from all seven species belonging to the two genera, Astrapia and Paradigalla. These species inhabit different montane regions across New Guinea but sometimes have overlapping ranges. We find multiple signals of historical gene flow between and within the two genera. Additionally, we present the first empirical genomic evidence of a hybrid zone between two lekking bird-of-paradise species. In the final chapter, chapter IV, we investigate whether introgressed variants may have contributed to the evolution of phenotypic traits in the genera Astrapia, Paradigalla, Epimachus and Drepanornis. We use window-based tests of treeness to pinpoint genomic regions with an excess of shared alleles and further investigate the genes present in these regions and their function. The overarching aim of this thesis was to study to what extent lekking birds-of-paradise still hybridise today, to what degree hybridisation can lead to introgression and whether introgression may facilitate phenotypic evolution. 

The family Melampittidae is endemic to New Guinea and consists of two monotypic genera: Melampitta lugubris (Lesser Melampitta) and Megalampitta gigantea (Greater Melampitta). Both Melampitta species have scattered and disconnected distributions across New Guinea in the central mountain range and in some of the outlying ranges. While M. lugubris is common and found in most montane regions of the island, M. gigantaea is elusive and known from only six localities in isolated pockets on New Guinea with very specific habitats of limestone and sinkholes. In this project, we apply museomics to determine the population structure and demographic history of these two species. We re-sequenced the genomes of all seven known M. gigantaea samples housed in museum collections as well as 24 M. lugubris samples from across its distribution. By comparing population structure between the two species, we investigate to what extent habitat dependence, such as in M. gigantaea, may affect population connectivity. Phylogenetic and population genomic analyses, as well as acoustic variation revealed that M. gigantaea consists of a single population in contrast to M. lugubris that shows much stronger population structure across the island. We suggest a recent collapse of M. gigantaea into its fragmented habitats as an explanation to its unexpected low diversity and lack of population structure. The deep genetic divergences between the M. lugubris populations on the Vogelkop region, in the western central range and the eastern central range, respectively, suggests that these three populations should be elevated to full species level. This work sheds new light on the mechanisms that have shaped the intriguing distribution of the two species within this family and is a prime example of the importance of museum collections for genomic studies of poorly known and rare species.

A debate in conservation genomics centers on whether to conserve small, fragmented populations independently or blend them through translocations from larger populations. Translocations of red grouse (Lagopus scotica) from Great Britain to supplement the Irish population have been suggested. We incorporate a variety of genetic datasets to address this. We used genome wide data from 23 contemporary and historic red grouse from Great Britain and Ireland. We also investigate microsatellite data, sequence candidate pigmentation genes, and assess phenotypic color variation. Genomic data indicate higher inbreeding in Irish grouse relative to an English population and significant divergence for genomic (FST = 0.095) and microsatellite (FST = 0.03) markers. Contemporary Ne was seven times smaller in the Irish population compared to the English. We identified divergent regions linked to pigmentation, immune response, and food intake. We show phenotypic differences in plumage color and sequence divergence among coding regions in the melanin pathway including MC1R (FST from genomic data of 0.3). The two populations thus appear locally adapted and this divergence between the source and target population when used for conservation translocations can swamp locally adapted alleles and/or introduce maladapted genotypes, leading to outbreeding depression. While it is important to avoid inbreeding by sustaining larger populations, our research emphasizes the need for practitioners to consider population divergence and local adaptation. We advocate against translocations between Ireland and Britain as a conservation strategy in this particular case and underscore the importance of prioritizing local populations where possible.

The aim of this study was to correlate plumage variation with the amount of genomic hybrid content in hybrids between Azure Tits Cyanistes cyanus (Pallas, 1770) and European Blue Tit Cyanistes caeruleus (Linnaeus, 1758), by re-sequencing the genomes of museum specimens of non-hybrids and presumed hybrids with varying plumages. The project was funded by crowdsourcing and initiated when two presumed Azure Tits, observed by hundreds of Swedish birdwatchers, were rejected as hybrids based on minor plumage deviations assumed to indicate hybrid contents from the European Blue Tit. The results confirm that hybrids with intermediate plumages, so called Pleske’s Tits, are first generation hybrids (F1 hybrids). Individuals, whose plumages are similar to Azure Tits, but assessed as hybrids based on minor plumage deviations, are all backcrosses but vary in their degree of hybrid content. However, some individuals morphologically recognized as pure Azure Tits expressed similar degrees of hybrid content. The results indicate that: (1) hybrid content may be widespread in Azure Tits in the western part of its habitat distribution; (2) plumage deviation in backcrosses is not linearly correlated with the genetic degree of hybrid origin; and (3) all Azure Tits observed in Europe outside its natural distribution may have some degree of hybrid origin. We therefore suggest that it is very difficult to phenotypically single out hybrids beyond first generation backcrosses. We argue that decreased sequencing costs and improved analytical tools open the doors for museomic crowd-sourced projects that may not address outstanding biological questions but have a major interest for lay citizens such as birdwatchers.

The authors used museomics to reconstruct the mitochondrial genome from two individuals of the Moroccan, endemic and extinct trout, Salmo pallaryi. They further obtained partial data from 21 nuclear genes previously used for trout phylogenetic analyses. Phylogenetic analyses, including publicly available data from the mitochondrial control region and the cytochrome b gene, and the 21 nuclear genes, place S. pallaryi among other North African trouts. mtDNA places S. pallaryi close to Salmo macrostigma within a single North African clade. Although the nuclear coverage of the genome was low, both specimens were independently positioned as sisters to one of two distantly related North African clades, viz. the Atlas clade with the Dades trout, Salmo multipunctatus. Phylogenetic discordance between mtDNA and nuclear DNA phylogenies is briefly discussed. As several specimens that were extracted failed to produce DNA of sufficient quality, the authors discuss potential reasons for the failure. They suggest that museum specimens in poor physical condition may be better for DNA extraction compared to better-preserved ones, possibly related to the innovation of formalin as a fixative before ethanol storage in the early 20th century.

Biological specimens in natural history collections constitute a massive repository of genetic information. Many specimens have been collected in areas in which they no longer exist or in areas where present-day collecting is not possible. There are also specimens in collections representing populations or species that have gone extinct. Furthermore, species or populations may have been sampled throughout an extensive time period, which is particularly valuable for studies of genetic change through time. With the advent of high-throughput sequencing, natural history museum resources have become accessible for genomic research. Consequently, these unique resources are increasingly being used across many fields of natural history. In this paper, we summarize our experiences of resequencing hundreds of genomes from historical avian museum specimens. We publish the protocols we have used and discuss the entire workflow from sampling and laboratory procedures, to the bioinformatic processing of historical specimen data.