The Mitogenome Relationships of Barn Swallows (Hirundo rustica)

The avian mitochondrial genome is organised in a small circular molecule of DNA, present in hundreds/thousands of copies per cell, and is transmitted without recombination via strictly maternal lines. This molecule is also characterised by a far greater evolutionary rate than the average nuclear gene, a rate which is especially faster for Neoaves. These characteristics make mitochondrial DNA (mtDNA) an important tool to study the history and phylogeny of a bird species, aided also by the sequential nature of accumulation of mutations which can be traced back along parallel monophyletic units (clades), called haplogroups, which tend to be generally restricted to specific geographical areas, population groups or subspecies after colonisation events. The combined study of the geographical distributions of populations within a species, their variability and the coalescent ages of the in the phylogeny is known as ‘phylogeography’. The timescale of these events is obtained by converting the genetic diversity between lineages to age estimates by using a molecular clock. The acquired ages together with geographical distribution information of mitochondrial DNA lineages allow for inference of the demographic history of the species such as dispersals, range expansions or migrations. The barn swallow (Hirundo rustica) is a model species for behavioural and ecological studies. It is a polytipic species consisting of several subspecies with a Holoarctic breeding distribution. These subspecies include the European barn swallow, H. r. rustica (Europe, North Africa and Western Asia), the Egyptian barn swallow, H. r. savignii (Egypt), the Levant barn swallow, H. r. transitiva (Israel, Lebanon, Jordan and Syria), the Tytler barn swallow, H. r. tytleri (southern-central Siberia, Mongolia), the Amur barn swallow, H. r. gutturalis (central-eastern China, Japan) and the American barn swallow, H. r. erythrogaster (North America). Previous studies have generally assessed only a rather limited portion of the barn swallow mtDNA, thus limiting the resolution of the phylogenies and, in turn, hindering the possibility to address relevant rising questions concerning subspecies relationships, genetic structure and demography. However, a much higher level of phylogenetic resolution can be readily achieved by sequencing entire mitogenomes, an approach that has been employed for many other animal species. During my three years of research, I employed next generation sequencing (NGS) to obtain 411 complete mitogenomes, mainly from the European H. r. rustica, but also from other subspecies. Phylogenetic and Bayesian analyses allowed me to (i) obtain a high-resolution mitogenome phylogeny of the species; (ii) define the matrilineal relationships between subspecies and their divergence times; and (iii) assess the species demography through time. This was done by testing different DNA extraction, purification and amplification methods and different NGS library preparation kits and sequencing cartridges (for the MiSeq) to substitute the previously used Sanger technology. In addition, given that passerines are so small, they are virtually absent from the fossil record which makes the calibration of a molecular clock an arduous task. To tackle this, I spent my final year concentrating on finding a suitable molecular clock specific for the barn swallow via bioinformatical means. This resulted in the first swallow complete mitogenome coding sequence-calibrated molecular clock. Taken together, the data and results reported in this thesis remark the fact that phylogeography analyses of mitogenomes remain an essential element to assess species origins and subspecies relationships in the context of an animal evolution perspective that is becoming increasingly multidisciplinary.The avian mitochondrial genome is organised in a small circular molecule of DNA, present in hundreds/thousands of copies per cell, and is transmitted without recombination via strictly maternal lines. This molecule is also characterised by a far greater evolutionary rate than the average nuclear gene, a rate which is especially faster for Neoaves. These characteristics make mitochondrial DNA (mtDNA) an important tool to study the history and phylogeny of a bird species, aided also by the sequential nature of accumulation of mutations which can be traced back along parallel monophyletic units (clades), called haplogroups, which tend to be generally restricted to specific geographical areas, population groups or subspecies after colonisation events. The combined study of the geographical distributions of populations within a species, their variability and the coalescent ages of the in the phylogeny is known as ‘phylogeography’. The timescale of these events is obtained by converting the genetic diversity between lineages to age estimates by using a molecular clock. The acquired ages together with geographical distribution information of mitochondrial DNA lineages allow for inference of the demographic history of the species such as dispersals, range expansions or migrations. The barn swallow (Hirundo rustica) is a model species for behavioural and ecological studies. It is a polytipic species consisting of several subspecies with a Holoarctic breeding distribution. These subspecies include the European barn swallow, H. r. rustica (Europe, North Africa and Western Asia), the Egyptian barn swallow, H. r. savignii (Egypt), the Levant barn swallow, H. r. transitiva (Israel, Lebanon, Jordan and Syria), the Tytler barn swallow, H. r. tytleri (southern-central Siberia, Mongolia), the Amur barn swallow, H. r. gutturalis (central-eastern China, Japan) and the American barn swallow, H. r. erythrogaster (North America). Previous studies have generally assessed only a rather limited portion of the barn swallow mtDNA, thus limiting the resolution of the phylogenies and, in turn, hindering the possibility to address relevant rising questions concerning subspecies relationships, genetic structure and demography. However, a much higher level of phylogenetic resolution can be readily achieved by sequencing entire mitogenomes, an approach that has been employed for many other animal species. During my three years of research, I employed next generation sequencing (NGS) to obtain 411 complete mitogenomes, mainly from the European H. r. rustica, but also from other subspecies. Phylogenetic and Bayesian analyses allowed me to (i) obtain a high-resolution mitogenome phylogeny of the species; (ii) define the matrilineal relationships between subspecies and their divergence times; and (iii) assess the species demography through time. This was done by testing different DNA extraction, purification and amplification methods and different NGS library preparation kits and sequencing cartridges (for the MiSeq) to substitute the previously used Sanger technology. In addition, given that passerines are so small, they are virtually absent from the fossil record which makes the calibration of a molecular clock an arduous task. To tackle this, I spent my final year concentrating on finding a suitable molecular clock specific for the barn swallow via bioinformatical means. This resulted in the first swallow complete mitogenome coding sequence-calibrated molecular clock. Taken together, the data and results reported in this thesis remark the fact that phylogeography analyses of mitogenomes remain an essential element to assess species origins and subspecies relationships in the context of an animal evolution perspective that is becoming increasingly multidisciplinary