This thesis examines the evolutionary divergence between northern European and Italian populations of Chorthippus parallelus. Several differing approaches were taken, which identified the inception of various components of the speciation process between these parapatric populations which meet in the Alps. Firstly, partial post-zygotic reproductive isolation was demonstrated using hybrid crosses. The male hybrid offspring of both reciprocal crosses were sterile, displaying severe testicular dysfunction, while the female hybrids showed no deleterious effects of hybridisation. In this grasshopper the males are the heterogametic sex, possessing a single X chromosome, and so this pattern of hybrid sterility conforms to Haldane's rule. Secondly, investigation of the calling song of the male grasshopper, a component of the mate recognition system, suggested the presence of pre-mating reproductive isolation. Males from the different races were found to sing calling song of a significantly different structure. Finally, examination of DNA sequence divergence in a mitochondrial DNA marker demonstrated Significant levels of genetic differentiation between the races. This population divergence and incipient reproductive isolation parallels that found between the north European and Iberian populations of this grasshopper, and provides further evidence that the divergent geographical races have resulted from allopatric divergence while in isolated refugial populations during the glacial periods of the Pleistocene Epoch. These approaches were repeated to investigate genetic divergence between localised populations within the Italian peninsula. No hybrid dysfunction was observed between these populations, suggesting that they are recently derived from one continuous population. This was probably the refugial population of the last ice-age. Additionally, investigation with the mtDNA marker gave preliminary evidence for population expansion from the south to the north of Italy. Interestingly, the male calling song was Significantly different in populations from the north and the south of Italy, suggesting that a component of pre-mating reproductive isolation may have evolved prior to post-mating isolation in allopatric populations of C. parallelus.

Ammomanes cinctura (Bar-tailed Lark) is a lark species with a wide distribution in the Palearctic. One of its subspecies, A. c. arenicolor, has a wide range across northern Africa, within which it shows very minor morphological variation but deep divergence in the mitochondrial cytochrome b locus between two geographically widely separated populations. There are two additional allopatric subspecies, A. c. cinctura (Cape Verde Islands) and A. c. zarudnyi (Iran to Pakistan), which differ slightly more in morphology. The genomic population structure, evolutionary history, and taxonomic status of the different populations within this species remain unclear. I applied an integrative approach, using genomic single nucleotide polymorphisms (SNPs), mtDNA and morphological data, to investigate the evolutionary divergence within Ammomanes cinctura. I acquired whole-genome sequence data from twelve individuals from Morocco (n=2), Saudi Arabia (n=7), and Iran (n=3), and performed phylogenetic and population structure analyses. Mitochondrial genomes were assembled and cytochrome b was extracted for phylogeny. Biometric measurements and quantified plumage analysis were conducted on museum specimens of all three subspecies. According to the mitochondrial data, the samples from Saudi Arabia and Iran form a clade that is deeply diverged (5.94 Mya, 95%HPD 3.15–8.95 Mya) from a clade comprising the samples from Morocco and Cape Verde Islands. In contrast, the nuclear SNPs recovered a very shallow divergence (0.095 Mya, 95%HPD 0.04-0.16 Mya) and weak population structure between the samples from Morocco vs. Saudi Arabia–Iran. Morphological results indicated that zarudnyi is slightly differentiated from the other two subspecies, with a larger body size, and the three subspecies are slightly divergent in plumage. The close similarity between Moroccan and Saudi Arabian birds in morphology was also confirmed, in conflict with the molecular data – highlighting the problem with trinomials in this case. The results suggest that the deep divergence in mitochondrial DNA is due to a complex evolutionary history.