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In birds, song can evolve quickly through cultural transmission and due to errors in the learning process may result in regional dialects. A lack of dialectal recognition may be a critical component of reproductive isolation through female mate preference. I investigated the role of cultural divergence in reproductive isolation in a widespread Neotropical passerine the rufous-collared sparrow (Zonotrichia capensis). In Chapter II, I investigated, the role of female preference for and male territorial response towards, local and non-local dialects in two allopatric populations. Females in both populations preferred their local dialect to the dialect of an allopatric population only 25 km away. In contrast, males showed similar territorial response to all conspecific dialects. Premating reproductive isolation based on culture may exist among the study populations. In chapter III, I investigated if cultural divergence can drive population divergence by examining dialects and variable microsatellite loci among eight populations of Z. capensis. I investigated the presence of population divergence and then identified the mechanism that may be driving the pattern. Apart from culture, a geographic barrier (the Andean ridge), elevation, and geographic distance were potential mechanisms of population divergence that I considered. All sites exhibited localized dialects, except for two closest neighboring populations. Populations exhibited genetic differentiation with support for the presence of five genetic clusters. Culture does not appear to be driving population divergence because song dialects and genetic population structure were not correlated. Populations separated by an Andean ridge did not display higher genetic or song differences than distance would predict, suggesting that the ridge is not driving population divergence. Elevation was not correlated to song or genetic differentiation. Both song divergence and microsatellite allele frequency differentiation were correlated with geographic distance suggesting a pattern of isolation by distance. Overall, geographic distance is the best predictor of population divergence in this system. Cumulatively, I found that culture might promote assortative mating via female mate choice, perhaps generating partial reproductive isolation; however, song dialect differences among contiguous populations is not currently driving population divergence in this species. / Ph. D.

The formation and persistence of species are the subject of much debate among biologists. Many species of Drosophila are behaviourally isolated, meaning that heterospecific individuals are not attracted to one another and do not interbreed. Often, this behavioural isolation is at least in part due to differences in pheromonal preference. Drosophila pheromones are long-chain cuticular hydrocarbons (CHCs). Desaturases are enzymes that are important for the production of CHCs. This thesis investigates the evolution of the gene family across 12 species of Drosophila. Desaturase genes were located in all species. Some genes, those that have previously been shown to have important roles in pheromonal communication, have experienced duplication and loss in several species. Two previously undiscovered duplicates were identified. Generally the desaturase gene family is governed by purifying selection, although following duplication these constraints are relaxed and in some cases duplicated genes show compelling evidence of positive selection. One of the loci under positive selection, the novel duplicate desat1b of the obscura group, was found to have a sex-biased expression pattern and alternative splicing in its 5′ UTR. In RNAi knock-down experiments of desaturase gene function in D. melanogaster, several desaturases were shown to affect CHC profiles of males and females, including some that were previously unlinked to CHC production.

Comment la diversification biologique peut-elle avoir lieu malgré les échanges génétiques? Comment les barrières reproductives entre espèces évoluent-elles et fonctionnent-elles? Les changements adaptatifs de certains traits favorisent-ils la diversification et la spéciation? Ces questions ouvertes en biologie évolutive constituent la base de ce projet. Pour y répondre, nous nous sommes intéressés aux papillons du genre néo-tropical Heliconius qui constituent une partie importante des communautés diversifiées de papillons néotropicaux. Les papillons de ce groupe sont immangeables pour les prédateurs, arborent des colorations d’avertissement qui signalent leur toxicité, et miment d’autres papillons toxiques dans leurs communautés locales. Ce genre a connu une radiation adaptative des motifs colorés soumis à la sélection naturelle favorisant le mimétisme de divers signaux locaux, mais ces motifs sont également connus comme signaux intraspécifiques favorisant les appariements homogames. Mes travaux ont permis d’approfondir les connaissances actuelles sur la fonction écologique et la base génétique de la couleur des ailes chez ces papillons, et d’explorer l'importance de la couleur des ailes par rapport aux signaux chimiques au cours des premières étapes de diversification. Dans cette optique, j’ai caractérisé la divergence adaptive entre les taxons à différents stades du continuum de spéciation, par une approche intégrative combinant des données génomiques, phénotypiques, comportementales, chimiques et écologiques. Plus précisément, j’ai étudié le sous-clade de Heliconius appelé sylvaniformes, contenant des espèces de papillons aux motifs tigrés, qui participent à des relations de mimétisme avec de nombreuses autres espèces fortement apparentées ou non. Mes travaux incluent la description comparative de l'architecture génétique des motifs colorés adaptatifs parallèlement chez les espèces H. hecale et H. ismenius, en utilisant des croisements, du génotypage génomique à haut débit, et de la morphométrie des motifs colorés. J’ai également exploré l'importance de la sélection naturelle et sexuelle sur les locus contrôlant ces motifs colorés aux stades précoces de divergence dans ce genre. En particulier, j’ai analysé la structure et le maintien de la zone d’hybridation entre deux races parapatriques de H. hecale montrant des colorations différentes, en combinant la génétique et la génomique des populations, ainsi que l’analyse phénotypique de clines et des tests comportementaux sur le choix de partenaire chez les mâles. Enfin, j’ai effectué des analyses génomiques de la divergence et du flux de gènes en me basant sur des données de re-séquençage de génomes complets afin de rechercher des traces d'introgression entre des espèces co-mimétiques et étroitement apparentées. Ceci a été également couplé à des expériences de préférence et de comportement sexuel, ainsi qu’à des analyses chimiques montrant d'importantes différences dans des composés qui pourraient intervenir dans la reconnaissance spécifique et le maintien des limites entre espèces. Dans l'ensemble, mes travaux montrent que bien que la sélection agissant sur les motifs colorés des ailes ait été centrale dans la diversification du genre Heliconius, l'accumulation d'autres barrières au flux de gènes peut jouer un rôle important dans l’aboutissement du processus de spéciation. / How does biological diversification occur in the face of genetic exchange? How do reproductive barriers evolve and function? What is the role of adaptive traits in promoting diversification and speciation? These open questions in evolutionary biology are at the core of this project. In order to tackle them, we have focused on butterflies in the neo-tropical genus which are an important component of the diverse butterfly communities in the Neo-tropics. Butterflies in the genus Heliconius are unpalatable to predators, use warning colours to advertise their defences, and mimic other defended butterflies in their local communities. The genus has undergone an adaptive radiation in wing colour patterns as a result of natural selection for mimicry, and is also well known for assortative mating based on wing pattern. I have extended the current knowledge about the ecological function and the genetic basis of wing color patterns in these butterflies and explored the importance of wing coloration relative to chemical signaling in the early stages of diversification. To this aim, I have characterised the adaptive divergence between lineages at different stages of the speciation continuum, by integrating genomic, phenotypic, behavioural, chemical and ecological data. More precisely, I have studied the so-called silvaniform sub-clade of Heliconius, known for harbouring species with tiger patterns that participate in mimicry with large groups of other closely and distantly-related species. My work includes the comparative description of the genetic architecture of wing pattern adaptation in two species, H. hecale and H. ismenius, using crosses, genome-wide next-generation genotyping, and advanced morphometrics of colour patterns. I have also explored the importance of natural and sexual selection on wing-patterning loci at early stages of divergence in the genus. In particular, I have analysed the structure and maintenance of a hybrid zone between two distinctly coloured parapatric races of H. hecale by using a combination of population genetics and genomics, coupled to a phenotypic analysis of the clines and to behavioural assays on male-based mate choice. Finally, I have carried out genome-wide analyses of divergence and gene flow with whole genome sequencing data to look for evidence of introgression between coexisting, hybridising co-mimetic species. This was again coupled to experiments on mating preferences and behavior, and yielded evidence for important differences in putative pheromone signals which may mediate species recognition and the maintenance of species boundaries. Overall, my results show that although selection on wing pattern divergence have been central to the diversification of the genus Heliconius, the accumulation of other barriers to gene flow may be important for the speciation process to be completed.

O percevejo-marrom-da-soja, Euschistus heros (F.) (Hemiptera: Pentatomidae), é uma praga chave da soja na América do Sul. Recentemente, foi descoberto a presença de duas linhagens alopátricas de E. heros no Brasil. Uma linhagem de ocorrência predominante na região Norte/Nordeste (N) e outra predominante na região Sul (S) e uma zona sobreposição entre as linhagens no Cerrado. O encontro de dois pools gênicos pode representar aumento ou diminuição de vigor das populações mediado pelo grau de isolamento reprodutivo. O encontro das linhagens no Cerrado é uma hipótese para as explosões populacionais de E. heros em cultivos de soja e constante ocorrência em cultivos de algodão relatados nos últimos anos. Questões sobre o potencial adaptativo dessas linhagens são de grande importância para entendimento da dinâmica e dos surtos populacionais de E. heros. Assim, nossos objetivos foram: 1) verificar a presença de barreiras reprodutivas entre as duas linhagens alopátricas de E. heros que promova uma prole híbrida de baixo fitness; e 2) avaliar o efeito da temperatura e fotoperíodo no tempo de desenvolvimento e viabilidade de fases imaturas e na expressão da dormência nas linhagens alopátricas de E. heros. Cruzamentos recíprocos foram realizados com intuito de avaliar o desenvolvimento da prole híbrida quando comparada com a prole coespecífica. Os parâmetros biológicos e de tabela de vida evidenciaram que o cruzamento entre as duas linhagens não representou diminuição de fitness da prole híbrida, portanto, não existe incompatibilidade pós-zigótica entre as linhagens de E. heros. Adicionalmente, ovos das linhagens (S e N) foram incubados em câmaras climáticas sob diferentes regimes de luz (10: 14, 11,5: 12,5 e 14: 10 (L: E)); e temperaturas (14,18, 21 e 25°C). Ficou evidente que existe uma relação entre temperatura e fotoperíodo no tempo de desenvolvimento e viabilidade de E. heros. A temperatura de 18°C e fotoperíodo de 10: 14 (L:E) aumentam o tempo necessário para completar o ciclo e reduzem a viabilidade. Temperatura de 25°C e fotoperíodo de 14: 10 (L:E) diminuem tempo de desenvolvimento e aumentam a viabilidade nas fases imaturas das duas linhagens. A expressão do fenótipo da dormência foi positivamente afetada por temperaturas menores e fotoperíodos mais curtos, especialmente para a linhagem Sul. A linhagem Norte não expressou características fenotípicas de dormência mesmo sob condições de temperaturas baixas e dias curtos. As diferenças encontradas possivelmente estão relacionadas ao local de origem das duas linhagens. Assim, o encontro de dois pools gênicos onde não há barreira reprodutiva aumenta a diversidade genética nas populações e permite a combinação de alelos que podem aumentar o vigor das populações e a capacidade de adaptação a ambientes novos e perturbados, como o ambiente agrícola. Os resultados demonstram que as duas linhagens possuem respostas similares aos fatores de estresses abióticos nas fases imaturas e distintas na expressão do fenótipo da dormência. As respostas deste trabalho oferecem informações úteis para melhor entender a dinâmica populacional contemporânea e para implementação de estratégias de manejo mais eficientes e sustentáveis de E. heros no cenário agrícola nacional. / The brown soybean stink bug, Euschistus heros (F.) (Hemiptera: Pentatomidae), is a key soybean pest in South America. It was recently discovered the presence of two allopatric lineages of E. heros in Brazil. A lineage predominantly occurring in the Northern/Northeast region (N) and another predominate in the Southern region (S) and an overlapping hybridization zone in the Cerrado region. The meeting of two gene pools may represent an increase or decrease in the vigor of populations in the absence of reproductive isolation. The reunion of the lineages in the Cerrado is a hypothesis for the expressive increase of the species in soybean cultivations and expansion of problems in the cotton crop. Questions about the adaptive potential of these lineages are of great importance for understanding the dynamics and population outbreaks of E. heros. The present study aims to: 1) verify the presence of reproductive barriers between the two lineages by means of a low fitness hybrid offspring; 2) to evaluate the effect of temperature and photoperiod on the development time and viability of immature phases and expression of dormancy in the allopatric lineages of E. heros. Reciprocal crosses were performed with the purpose of evaluating the development of the hybrid offspring when compared to the co-specific offspring. The biological and life table parameters showed that the crossbreeding between the two lineages did not represent a decrease in the fitness of the hybrid offspring. Therefore, there is no post-zygotic incompatibility among the lineages of E. heros. Additionally, eggs from both lineages (S and N) were incubated in climatic chambers under different light regimes (10: 14, 11,5: 12,5 and 14: 10 (L: E) and temperatures (14, 18, 21 and 25°C). It was evident that there is a relationship between temperature and photoperiod in the development time and viability of E. heros. The temperature of 18°C and photoperiod of 10: 14 (L: E) increase the time required to complete the cycle and reduce viability. Temperature of 25°C and photoperiod of 14: 10 (L: E) decrease development time and increase viability in the immature phases of the two lineages. The expression of the dormancy phenotype was positively affected by shorter temperatures and shorter photoperiods, especially for the Southern lineage. The Northern lineage did not express phenotypic characteristics of dormancy even under low temperature and short-day conditions. The differences found are possibly related to the geographical origin of the two lineages. Thus, the meeting of two gene pools where there is no reproductive barrier increases the genetic diversity in populations and, allows the combination of alleles that can increase the vigor of populations and the ability to adapt to new and disturbed environments, such as the agricultural environment. The results demonstrate that the two strains have similar responses to the abiotic stress factors in the immature phases and distinct in the expression of the dormancy phenotype. The answers of this work offer useful information to better understand contemporary population dynamics and to implement more efficient and sustainable management strategies of E. heros in the national agricultural scenario.

This thesis investigates aspects of the ecology, morphology, bioacoustics, genetic relatedness, biogeography and taxonomy of cicadas in the Pauropsalta annulata Goding and Froggatt species complex, across their entire eastern Australian geographical distribution. These cicadas seem to emerge at the same sites annually, although the exact duration of their life cycle is not known. They are wary and mobile insects, with individuals generally taking flight at the first sign of disturbance, which makes them challenging to record, capture and observe. The males produce distinctive calling songs that have a consistent rhythmic structure, which is critically important for attracting females. In most cases, the songs are biphasic, with a penetrating introductory or "buzzing" component that apparently functions in mate attraction and a strongly structured cueing or "lilting" component for mate localisation. Initially Pauropsalta annulata was thought to comprise a single species with some degree of geographically structured variation in its calling song. Consequently, various "song types" had been recognised, but their precise relationship to one another had never been investigated. Therefore the structure of their calling songs were compared statistically across individuals of three song types, and this revealed four discrete clusters that were demonstrated to be independent of one another and consistent in their calling song structure. Plotting the geographical distributions of these cicadas revealed that each of the P. annulata song types is independently distributed geographically, with areas of overlap that are relatively small. Calling song structure is consistent for each song type across extensive geographical space and this structure holds even into areas of sympatry. One song type showed consistent differences from the others in male genitalia structure, and female ovipositor length differs significantly among three of the other song types. Each song type was found to be strongly associated with a small number of tree species and these associations are maintained into areas of sympatry. The spatial ecological and morphological comparisons in song structure, plant associations and morphology made in this study demonstrate that the P. annulata song types actually represent a complex of cryptic species. Two of the song types appear to intergrade in areas of sympatric overlap in terms of calling songs, plant associations and morphology, and thus represent subspecies as defined in population genetics terms. The components of the male calling song were then investigated to determine their specific functions and thus how they could delimit species boundaries. Significant differences were found in dominant song frequency between three of the four species, and may contribute to differential mate attraction. Both components of the song of each species have the same dominant frequency. Consistent differences were also evident, among species and subspecies, in the rhythmic structure of the "lilting" component of the male calling song. This component contains repeated phrases and each one of these provides a cue to which the conspecific female may respond. Her response is timed for the brief silent interval between the phrases. At this point the calling song becomes a duet, which enables the male to locate the female, as he actively searches for her on the surrounding branches at this stage. The calling song is discussed in terms of random mating within gene pools of these cicadas and in terms of its role as part of their broader fertilisation mechanism. A molecular analysis of the P. annulata species complex was performed to examine the phylogenetic relationships across 12 species and four subspecies defined in this study, and estimate divergence times within the group. Individual specimens were sampled widely across the geographical distributions of the species and subspecies where possible to account for genetic variation across space. DNA sequences from two loci were amplified: mitochondrial CO1 ("barcoding region") and a large intron from the dynamin nuclear gene. Separate phylogenies were reconstructed for each locus using maximum parsimony procedures and Bayesian posterior sampling with implementation of a relaxed molecular clock. The phylogenies from both genes provided strong support for the monophyly of the P. annulata species complex, and nine of the species were monophyletic based on the CO1 gene. The remaining three emerged non-monophyletic. Based on a clock calibration of 0.0165s/s/myr, the monophyletic clades represented by extant P. annulata species diverged about 4.5-8.0 million years ago. Those species that emerged non-monophyletic had shallower divergences, with the exception of one species, which exhibited haplotype diversity that conferred up to 13.2% sequence divergence between allopatric populations in CO1. Dynamin produced a broadly similar phylogenetic pattern to that of CO1, but the relationships among individuals across the species and subspecies that emerged non-monophyletic differed substantially. This lack of congruence between the two genomes, in combination with the dominance of internal haplotypes in both loci, indicates an overall pattern of deep coalescence rather than interspecific hybridization. Therefore the molecular data do not provide an alternative definition of species limits in the P. annulata species complex, despite some emerging non-monophyletic in this analysis. Sound interpretation of the phylogenetic pattern discovered here would not have been possible without the acoustic, ecological and geographical investigations on species limits that preceded this work. To determine what biological and climatic factors influence the present day distributions of these cicadas, the distributions of two of the most closely related species in the P. annulata species complex were compared with the distributions of the tree species with which each is mainly associated. Because a large part of their life cycle is subterranean, soil texture, pH, electrical conductivity and force required for surface penetration were compared across sites where each of the cicadas occur in sympatry and allopatry. Finally, the influence of temperature and rainfall variables were investigated by testing 'predicted distribution' models (formed using positive distribution records) against negative records for both sets of variables, individually and in combination. The results show that the extent of the distribution of the cicada species is substantially less than that of the associated tree species. The geographical distributions of one of the species may be influenced more by rainfall, or a combination of temperature and rainfall, whereas the other species appears to be more influenced by temperature alone. Both species tolerate soils with a wide range of pH levels, electrical conductivity and forces required for surface penetration. They both showed a strong association with soils that had a silt loam texture, with only few records from sandy soils. However, none of the soils sampled where the cicadas occurred were heavy clays, which suggests that the physical properties of such soils may provide an unsuitable environment for the nymphal stages of the life cycle of these particular cicadas. The resolution of species limits within the P. annulata species complex allowed the redescription of Pauropsalta annulata Goding and Froggatt sensu stricto and the description of 11 new species belonging to the P. annulata species group, all from eastern Australia. Two of these species comprise two subspecies each, also all new. New distribution records and calling song data are documented for the allied species, P. ayrensis Ewart, which is redescribed to include the characters newly recognised in the present study as significant taxonomically with respect to Pauropsalta cicadas. The treatment includes comprehensive descriptions of the morphology and calling songs of the species and subspecies, and separate descriptive keys are provided for both sets of characters. The new taxa comprise P. artatus sp. nov., P. corymbiae sp. nov., P. decorus sp. nov., P. graniticus sp. nov., P. inversus inversus subsp. nov., P. i. laboris subsp. nov., P. notialis notialis subsp. nov., P. notialis incitatus subsp. nov., P. simplex sp. nov., P. subtropicus sp. nov, P. torrensis sp. nov. and P. tremulus sp. nov. Areas of hybridization between P. n. notialis subsp. nov. and P. n. incitatus subsp. nov. are also documented, together with their calling songs and morphology, which justifies their subspecific status. The P. inversus subspecies are allopatric, but consistently differ in the duration between phrases of the calling song. Finally, the results and conclusions are amalgamated into a critical reassessment of what defines species limits and the most appropriate approaches to investigating species limits in sexual organisms. Some historical discussions are revisited, such as the question of the reality of species and how species are perceived under the premises of neoDarwinism. The realism of species demands that species limits are most realistically defined in terms of their fertilisation mechanism, for this delimits the gene pool and thus the distribution of adaptations (the calling song of cicadas, for example).

<p>In this thesis, different genetic tools are used to investigate pre- and postzygotic barriers to gene exchange and their role in speciation in the pied flycatcher (<i>Ficedula hypoleuca</i>) and the collared flycatcher (<i>F. albicollis</i>). This species complex consists of four genetically distinct clades that apparently diverged in allopatry (I). Sequencing of introns from autosomal and Z-linked genes from the two species reveals signs of selection on the Z-chromosome. Sexual selection acting on Z-linked genes might explain this pattern (II). By using large-scale genotyping of single nucleotide polymorphisms (SNPs), introgression is observed at autosomal- but not Z-linked loci, mostly from the pied- to the collared flycatcher. Male plumage characters and genes involved in hybrid fitness are largely mapped to the Z-chromosome (III). By studying mate choice of female hybrids I show that there is a link between female preferences and the Z chromosome (IV). The rate of introgression in island versus clinal hybrid zones is consistent with regional differences in hybrid fertility. Asymmetric gene flow from allopatry on the islands may oppose reinforcement, leading to introgression and a partial breakdown of postzygotic isolation. Adaptive introgression may explain the high rate of introgression observed at one of the genetic markers (V). For late breeding female collared flycatchers it appears to be adaptive to use pied flycatchers as social fathers but conspecific males as genetic fathers. Additionally, females in mixed species pairs may reduce hybridization costs by producing an excess of male hybrid offspring that are more fertile than females (VI).</p><p>In conclusion, the Z-chromosome appears to play a major role in flycatcher speciation. Sexual selection and reinforcement are important mechanisms in the divergence of these birds. However, gene flow from allopatry, introgression of adaptive genes and adaptive hetrospecific pairing by late breeding collared flycatcher females may work in the opposite direction.</p>

There is considerable evidence that pollinator shifts can explain many differences in flower morphology between closely related plant species, but the extent to which pollinator shifts can explain the maintenance of among-population variation in floral traits within species is poorly known. In this thesis, I combined comparative and experimental approaches to examine the evolution of floral traits in the moth-pollinated orchid Platanthera bifolia. More specifically, I investigated (1) the relationship between flower and pollinator morphology, (2) phenotypic selection on morphology and phenology in populations in contrasting environments, (3) components of prezygotic reproductive isolation among divergent populations, and (4) the adaptive and functional significance of two correlated floral traits. A study of Scandinavian of P. bifolia populations revealed that spur length was positively correlated with proboscis length of local pollinators, which suggests that variation in spur length reflects adaptive evolution in response to geographically variable pollinator-mediated selection. A phenotypic selection study on Öland, SE Sweden, suggested that disruptive selection on spur length contributes to the maintenance of a bimodal distribution of spur length in mixed habitats, but provided very limited evidence of divergent selection on plant morphology and flowering phenology in grassland and woodland habitats. Field experiments revealed strong reproductive isolation between divergent populations on Öland, due to differences in spatial distribution, flowering phenology, and pollinators, and among-population incompatibility. The results suggest that prezygotic reproductive isolation contributes to the maintenance of population differentiation in floral traits in P. bifolia. A field manipulation experiment demonstrated that spur length but not perianth size affects pollination success and seed production. This suggests that among-population differentiation in perianth size may be the result of a genetic correlation with spur length. Taken together, the results of this thesis suggest that pollinator-mediated selection can shape the evolution of intraspecific floral variation.

In this thesis, different genetic tools are used to investigate pre- and postzygotic barriers to gene exchange and their role in speciation in the pied flycatcher (Ficedula hypoleuca) and the collared flycatcher (F. albicollis). This species complex consists of four genetically distinct clades that apparently diverged in allopatry (I). Sequencing of introns from autosomal and Z-linked genes from the two species reveals signs of selection on the Z-chromosome. Sexual selection acting on Z-linked genes might explain this pattern (II). By using large-scale genotyping of single nucleotide polymorphisms (SNPs), introgression is observed at autosomal- but not Z-linked loci, mostly from the pied- to the collared flycatcher. Male plumage characters and genes involved in hybrid fitness are largely mapped to the Z-chromosome (III). By studying mate choice of female hybrids I show that there is a link between female preferences and the Z chromosome (IV). The rate of introgression in island versus clinal hybrid zones is consistent with regional differences in hybrid fertility. Asymmetric gene flow from allopatry on the islands may oppose reinforcement, leading to introgression and a partial breakdown of postzygotic isolation. Adaptive introgression may explain the high rate of introgression observed at one of the genetic markers (V). For late breeding female collared flycatchers it appears to be adaptive to use pied flycatchers as social fathers but conspecific males as genetic fathers. Additionally, females in mixed species pairs may reduce hybridization costs by producing an excess of male hybrid offspring that are more fertile than females (VI). In conclusion, the Z-chromosome appears to play a major role in flycatcher speciation. Sexual selection and reinforcement are important mechanisms in the divergence of these birds. However, gene flow from allopatry, introgression of adaptive genes and adaptive hetrospecific pairing by late breeding collared flycatcher females may work in the opposite direction.

This thesis investigates aspects of the ecology, morphology, bioacoustics, genetic relatedness, biogeography and taxonomy of cicadas in the Pauropsalta annulata Goding and Froggatt species complex, across their entire eastern Australian geographical distribution. These cicadas seem to emerge at the same sites annually, although the exact duration of their life cycle is not known. They are wary and mobile insects, with individuals generally taking flight at the first sign of disturbance, which makes them challenging to record, capture and observe. The males produce distinctive calling songs that have a consistent rhythmic structure, which is critically important for attracting females. In most cases, the songs are biphasic, with a penetrating introductory or "buzzing" component that apparently functions in mate attraction and a strongly structured cueing or "lilting" component for mate localisation. Initially Pauropsalta annulata was thought to comprise a single species with some degree of geographically structured variation in its calling song. Consequently, various "song types" had been recognised, but their precise relationship to one another had never been investigated. Therefore the structure of their calling songs were compared statistically across individuals of three song types, and this revealed four discrete clusters that were demonstrated to be independent of one another and consistent in their calling song structure. Plotting the geographical distributions of these cicadas revealed that each of the P. annulata song types is independently distributed geographically, with areas of overlap that are relatively small. Calling song structure is consistent for each song type across extensive geographical space and this structure holds even into areas of sympatry. One song type showed consistent differences from the others in male genitalia structure, and female ovipositor length differs significantly among three of the other song types. Each song type was found to be strongly associated with a small number of tree species and these associations are maintained into areas of sympatry. The spatial ecological and morphological comparisons in song structure, plant associations and morphology made in this study demonstrate that the P. annulata song types actually represent a complex of cryptic species. Two of the song types appear to intergrade in areas of sympatric overlap in terms of calling songs, plant associations and morphology, and thus represent subspecies as defined in population genetics terms. The components of the male calling song were then investigated to determine their specific functions and thus how they could delimit species boundaries. Significant differences were found in dominant song frequency between three of the four species, and may contribute to differential mate attraction. Both components of the song of each species have the same dominant frequency. Consistent differences were also evident, among species and subspecies, in the rhythmic structure of the "lilting" component of the male calling song. This component contains repeated phrases and each one of these provides a cue to which the conspecific female may respond. Her response is timed for the brief silent interval between the phrases. At this point the calling song becomes a duet, which enables the male to locate the female, as he actively searches for her on the surrounding branches at this stage. The calling song is discussed in terms of random mating within gene pools of these cicadas and in terms of its role as part of their broader fertilisation mechanism. A molecular analysis of the P. annulata species complex was performed to examine the phylogenetic relationships across 12 species and four subspecies defined in this study, and estimate divergence times within the group. Individual specimens were sampled widely across the geographical distributions of the species and subspecies where possible to account for genetic variation across space. DNA sequences from two loci were amplified: mitochondrial CO1 ("barcoding region") and a large intron from the dynamin nuclear gene. Separate phylogenies were reconstructed for each locus using maximum parsimony procedures and Bayesian posterior sampling with implementation of a relaxed molecular clock. The phylogenies from both genes provided strong support for the monophyly of the P. annulata species complex, and nine of the species were monophyletic based on the CO1 gene. The remaining three emerged non-monophyletic. Based on a clock calibration of 0.0165s/s/myr, the monophyletic clades represented by extant P. annulata species diverged about 4.5-8.0 million years ago. Those species that emerged non-monophyletic had shallower divergences, with the exception of one species, which exhibited haplotype diversity that conferred up to 13.2% sequence divergence between allopatric populations in CO1. Dynamin produced a broadly similar phylogenetic pattern to that of CO1, but the relationships among individuals across the species and subspecies that emerged non-monophyletic differed substantially. This lack of congruence between the two genomes, in combination with the dominance of internal haplotypes in both loci, indicates an overall pattern of deep coalescence rather than interspecific hybridization. Therefore the molecular data do not provide an alternative definition of species limits in the P. annulata species complex, despite some emerging non-monophyletic in this analysis. Sound interpretation of the phylogenetic pattern discovered here would not have been possible without the acoustic, ecological and geographical investigations on species limits that preceded this work. To determine what biological and climatic factors influence the present day distributions of these cicadas, the distributions of two of the most closely related species in the P. annulata species complex were compared with the distributions of the tree species with which each is mainly associated. Because a large part of their life cycle is subterranean, soil texture, pH, electrical conductivity and force required for surface penetration were compared across sites where each of the cicadas occur in sympatry and allopatry. Finally, the influence of temperature and rainfall variables were investigated by testing 'predicted distribution' models (formed using positive distribution records) against negative records for both sets of variables, individually and in combination. The results show that the extent of the distribution of the cicada species is substantially less than that of the associated tree species. The geographical distributions of one of the species may be influenced more by rainfall, or a combination of temperature and rainfall, whereas the other species appears to be more influenced by temperature alone. Both species tolerate soils with a wide range of pH levels, electrical conductivity and forces required for surface penetration. They both showed a strong association with soils that had a silt loam texture, with only few records from sandy soils. However, none of the soils sampled where the cicadas occurred were heavy clays, which suggests that the physical properties of such soils may provide an unsuitable environment for the nymphal stages of the life cycle of these particular cicadas. The resolution of species limits within the P. annulata species complex allowed the redescription of Pauropsalta annulata Goding and Froggatt sensu stricto and the description of 11 new species belonging to the P. annulata species group, all from eastern Australia. Two of these species comprise two subspecies each, also all new. New distribution records and calling song data are documented for the allied species, P. ayrensis Ewart, which is redescribed to include the characters newly recognised in the present study as significant taxonomically with respect to Pauropsalta cicadas. The treatment includes comprehensive descriptions of the morphology and calling songs of the species and subspecies, and separate descriptive keys are provided for both sets of characters. The new taxa comprise P. artatus sp. nov., P. corymbiae sp. nov., P. decorus sp. nov., P. graniticus sp. nov., P. inversus inversus subsp. nov., P. i. laboris subsp. nov., P. notialis notialis subsp. nov., P. notialis incitatus subsp. nov., P. simplex sp. nov., P. subtropicus sp. nov, P. torrensis sp. nov. and P. tremulus sp. nov. Areas of hybridization between P. n. notialis subsp. nov. and P. n. incitatus subsp. nov. are also documented, together with their calling songs and morphology, which justifies their subspecific status. The P. inversus subspecies are allopatric, but consistently differ in the duration between phrases of the calling song. Finally, the results and conclusions are amalgamated into a critical reassessment of what defines species limits and the most appropriate approaches to investigating species limits in sexual organisms. Some historical discussions are revisited, such as the question of the reality of species and how species are perceived under the premises of neoDarwinism. The realism of species demands that species limits are most realistically defined in terms of their fertilisation mechanism, for this delimits the gene pool and thus the distribution of adaptations (the calling song of cicadas, for example).