Sexual, or behavioral, isolation is a form of reproductive isolation that impedes gene flow between divergent taxa. In this study, sexual isolation was measured in a sister-species pair of North American cactophilic fruitflies. The results show that interspecific behavioral isolation is dependent upon the strain of origin of D. mojavensis, but is independent of the strain of origin of D. arizonae. Interspecific sexual isolation is greatest in crosses involving D. mojavensis from sympatry, which is consistent with reinforcement, and seems to be attributable to increased reluctance by D. mojavensis females and D. arizonae males to mate with heterospecifics. Contrary to previous studies, I found strong evidence for reproductive isolation between two strains of D. arizonae. The majority of the observed behavioral isolation seems to be the result of intraspecific, interpopulation encounters between females from Southeastern Mexico and males derived from the Northern population.

Doctor of Philosophy / Genetics Interdepartmental Program - Biology / Susan J. Brown / Understanding evolutionary processes that structure genetic variation associated with lineage diversification and speciation is a central problem. Shifts in host plant use by insect herbivores and subsequent divergence in mating signals can lead to sexual isolation, especially when selection for specialization on different hosts restricts gene flow among populations. The grasshopper Hesperotettix viridis (Thomas) is an oligophagous grasshopper feeding on plants primarily in the host plant genera Gutierrezia and Solidago in Kansas. I used mitochondrial and microsatellite genetic markers to evaluate the diversification pattern and underlying evolutionary mechanisms of two putative host races of H. viridis. I also quantified host preferences, the degree of sexual isolation among putative host races and divergence in cuticular attributes to identify the nature and origins of initial barriers that isolated populations in the formative stages of divergence. mtDNA data revealed a star-shaped phylogeny, suggesting isolation in a single refugium ~110,000 years ago based on a molecular clock, followed by rapid population expansion. Microsatellite data reveal significant host-based genetic differentiation and structuring in H. viridis populations in Kansas, including a microsatellite locus under strong divergent selection. Neutral microsatellite loci did not reveal a differentiation pattern specific to host plant use. Significant host-based preferences by individuals that fed on two host plant groups were detected in host paired-feeding preference studies. No-choice mate selection experiments revealed preferences for individuals collected from the same host species independent of location with little mating observed between individuals from different host species. Significant differentiation in color and cuticular composition among different host plant races within the study area was also detected. Correlations between host choice, mate choice and phenotypic divergence were observed and this host associated divergence appears to have a genetic basis. Based on the results of this study, I conclude that divergent selection for host plant use underlies observed sexual isolation among populations in this species. Hesperotettix viridis populations in Kansas that fed on Solidago and Gutierrezia species represent two incipient host races, early stages of diversification that could lead to speciation in insect herbivores.

Il existe chez D. melanogaster et D. simulans un polymorphisme des hydrocarbures mâles qui pourrait jouer un rôle dans la mise en place d’un isolement sexuel. Le 7-tricosène (7-T; C23:1) et le 7-pentacosène (7-P; C25:1) sont les phéromones principales des mâles. Elles interviennent dans le comportement de cour et varient en fonction des paramètres géoclimatiques. Cette thèse a pour objectif d'étudier le rôle des phéromones mâles sur l’isolement sexuel ainsi que les changements génétiques à l’origine de leur variation et de leur évolution. Un des volets de cette thèse porte sur des aspects physiologiques et comportementaux. Nous avons étudié l'impact de la température sur la synthèse des hydrocarbures mâles et l'influence du rapport 7-T/7-P sur la résistance à la dessiccation et sur la réceptivité des femelles de différentes populations de D. melanogaster et D. simulans. Nous avons observé un isolement sexuel significatif entre des souches 7-T et 7-P de ces deux espèces présentes au laboratoire depuis des décennies, ainsi qu'entre des lignées issues d'une même population soumises à une sélection artificielle. Il apparaît également que les souches synthétisant de grandes quantités de 7-P s’adaptent plus rapidement aux modifications importantes de température. La seconde partie de cette thèse consiste en la détermination et l'étude des gènes d’élongase pouvant être impliqués dans la synthèse du 7-T et du 7-P chez les mâles de D. melanogaster et dans une moindre mesure chez ceux de D. simulans. Les travaux réalisés nous ont permis de mettre en évidence qu’un gène, situé sur le chromosome II, joue un rôle majoritaire dans la synthèse du 7-P. / In D. melanogaster and D. simulans there is a male hydrocarbon polymorphism that may play a role in sexual isolation. The main male pheromones are the 7-tricosene (7-T C23: 1) and the 7-pentacosene (7-P, C25: 1). They are involved in courtship behavior and depend on geo-climatic parameters. The aim of this thesis is to study the role of male pheromones on sexual isolation and to better understand the genetic changes responsible for the variation and the evolution of male pheromones. The first part investigates the plasticity of CHCs in response to temperature and focuses on the role of 7-T and 7-P in resistance to desiccation and in sexual selection in D. melanogaster and D. simulans. In both species there was partial sexual isolation between 7-T and 7-P flies from wild-type laboratory strains and also from lines that have been artificially selected. Males with high levels of 7-P seemed to modify their CHCs profiles more quickly in response to temperature changes. The second part is the identification of elongase genes that might be involved in the synthesis of 7-T and 7-P in D. melanogaster and D. simulans males. A gene, located on chromosome II, could play a major role in the synthesis of 7-P in D. melanogaster.

Sexual imprinting, the process of learning mate preferences at a young age, could promote speciation by reducing attraction to individuals from divergent populations or species, consequently creating or maintaining reproductive isolation. Yet, despite the documentation of sexual imprinting in many taxa, its connection to speciation has been understudied. I chose to explore the potential link between sexual imprinting and reproductive isolation and in two North American rodents--the white-footed mouse (Peromyscus leucopus) and its sister species, the cotton mouse (Peromyscus gossypinus). These species have overlapping distributions in nature, possibly allowing interbreeding and admixture. In Chapter 1, I used double-digest restriction-associated DNA sequencing to test for hybridization in sympatric natural populations and found that 1.5% of sampled individuals showed evidence of admixture yet the species have maintained genetic distinctness in sympatry. In the lab, the species hybridize when given no choice of mates but mate more readily with conspecifics, suggesting that mating preferences may prevent hybridization in the wild. In Chapter 2, I tested whether mating preferences create significant reproductive isolation. I measured mating preferences in controlled laboratory conditions and found that both species and sexes preferred conspecific to heterospecific mates in 85% of trials. I then raised offspring with foster parents of the opposite species and found that P. leucopus has a genetically-determined preference while P. gossypinus learns its preference. In Chapter 3, I tested whether sexual imprinting on parental diet could generate assortative mating within a species. I tested this hypothesis by feeding P. gossypinus parents either orange- or garlic-flavored water, thereby exposing their offspring to these flavors through their parents until weaning. I tested the preferences of these offspring as adults and found that P. gossypinus, especially females, had strong assortative mating preferences. This implies that at least females learn parental dietary information and that assortative mating could evolve within a single generation. Together, my results confirm that sexual imprinting on parental traits--possibly mediated through dietary differences--can create assortative mating capable of generating sexual isolation and reducing gene flow between species. My research supports the importance of mating preferences and learning in speciation.

This study is primarily concerned with assessing the influence of the sex determination genes, transformer (tra), doublesex (dsx) and fruitless (fru) on three sexually dimorphic traits within Drosophila; pheromone blend, courtship song and sex comb tooth number. The sex determination loci have all been implicated as possible candidate genes affecting these important traits that contribute to sexual isolation, which is a major cause of speciation. Quantitative Trait Loci (QTL) analysis is used to assess the effects of these known candidate genes on the naturally occurring variation of mean interpulse interval (IPI) of courtship song and the differing pheromone blend profiles between Drosophila simulans and D. sechellia. The QTL analysis for both song and pheromone blend variation incorporated Multiple Interval Mapping (MIM), which enables the detection for epistasis. The desaturase loci desat1, desat2 and desatF were also included in the assessment on pheromone blends (cuticular hydrocarbon compounds), since they facilitate ecological adaptation and are also candidate genes, which are likely to exert a large affect on this particular trait. The sex determination genes were not significantly influential on the interspecific variation of the cuticular hydrocarbon compounds between these two sibling species. However significant effects were detected from two of the desaturase loci. desat1 was associated with a strong effect on the interspecific variation of a saturated hydrocarbon chain compound (unbranched-23). Additionally the candidate gene desatF potentially exerts an influence on the variation of 7,11-heptacosadiene, through a large epistatic effect with unidentified loci, situated between the markers pros and Mtn. The candidate gene eloF is situated in this region, and is known to affect the elongation of unsaturated hydrocarbon chains. The QTL associated with the marker desatF influenced the variation of both diene compounds (7,11-heptacosadiene and 7,11-pentacosadiene). Intriguingly epistasis was only detected for the variation of these two diene compounds. The MIM analysis assessing the affects of the sex determination genes on interspecific variation of mean IPI detected the candidate gene fru as the closest marker associated with a significant QTL on the third chromosome. The MIM also found a significant QTL associated with the marker Dgα situated on the second chromosome. Moreover significant epistatic interactions were detected between a further QTL situated nearest the marker forked on the X-chromosome with both of the other significant QTL situated on the third and second chromosomes. The analysis of a number of Recombinant Inbred (RI) lines was also carried out to test for the affects of the sex determination genes on both mean IPI and sex comb tooth number. The fru locus was associated with a significant increase in mean IPI, whereas the opposite was true for the dsx locus. In the analysis of sex comb tooth variation, it appears that all RI lines homozygous for D. sechellia alleles at the sex determination loci had significantly higher numbers of sex comb teeth. The final data chapter involves the sequence analysis of the fruitless locus, including all 13 fru proteins between ten recently sequenced Drosophilid genomes. The PAML program was used to detect the possible influence of natural selection on sequence divergence. There was no significant positive selection detected at the BTB functional domain and the sequences encoding for this domain were extremely conserved. Positive selection was found to be acting on the exon encoding for the Zinc-finger C domain. This domain is present in two protein isoforms including the male sex-specific isoform FRUMC, and the common non-sex-specific isoform FRUComC. Interestingly positive selection was also found at the non sex-specific Zinc-finger D domain.

ABSTRACT A major focus in biology is understanding the diversification of life and the processes that cause it. Much of this diversity is in the form of phenotypic variation among populations and species. In this thesis, I investigate two separate aspects of such phenotypic divergence. The first is the divergence of male mate preferences and their potential contribution to precopulatory sexual isolation and speciation. The second is the divergence of postcopulatory phenotypic divergence in the form of seminal fluid protein expression. With respect to the first aspect, in two separate experiments I investigated the contribution of male mate preferences to sexual isolation between two closely related fruit fly species experiencing differential costs to hybridization, Drosophila recens and Drosophila subquinaria. Male mate preferences are of particular interest because of their potential contribution to sexual isolation, a form of reproductive isolation which can contribute to speciation in sexually reproducing species. In the first experiment, I test for the presence of male mate preferences in each of the two species and whether the relative strength of the preference is concordant with the cost of hybridization. I found that that D. subquinaria males indiscriminately courted both their own (i.e. homospecific) females and heterospecific D. recens females. While D. recens from allopatry showed a similar pattern, those from sympatry courted their own females more than heterospecific females, indicating a pattern of reproductive character displacement. In the second experiment I test the role of learning in the context of these male mate preference in D. recens, and whether learning also showed a pattern of reproductive characteristic. I did not find evidence of learning in that D. recens males did not reduce their courting intensity towards heterospecific females after experiencing rejection by similar females. Consequently, I did not find an indication of reproductive character displacement. Finally, with respect to postcopulatory phenotypic divergence, I studied differences in seminal fluid protein expression between experimental populations of D. melanogaster experiencing one of three mating environments allowing for differing opportunities of mate competition and the environment in which it took place. These three mating environments include one in which mate competition was absent (MCabsent,), one in which mate competition occurred in a small, structurally simple environment (MCsimple), and one in which mate competition occurred in a larger, somewhat more complex environment (MCcomplex,). Male seminal fluids are of particular interest due to their ability to mediate postcopulatory competition between males and, therefore, can be used to manipulate females to a male’s own fitness benefit, potentially at her expense (i.e. sexual conflict). I investigated divergence in one particular seminal fluid protein implicated in sexual conflict, sex peptide (Acp70A). Whereas, gene expression levels among males from the three-mating treatment did not differ on average, relative stored quantities did, with MCcomplex males carrying significantly less sex peptide than either of MCabsent or MCsimple males (which did not differ from one another). This result suggests that mate competition and the environment in which it occurs play a significant role in the divergence of sex peptide phenotypes. ABSTRAIT Un objectif majeur de la biologie est de comprendre la diversification de la vie et les processus qui la provoquent. Une grande partie de cette diversité se présente sous la forme de variations phénotypiques entre les populations et les espèces. Dans cette thèse, j'étudie deux aspects distincts d'une telle divergence phénotypique. Le premier est la divergence des préférences des mâles et leurs contributions potentielles à l'isolement sexuel pré-copulatoire et à la spéciation. Le second est la différence de la divergence phénotypique post-copulatoire sous la forme de l'expression des protéines du liquide séminal. En ce qui concerne le premier aspect, dans deux expériences distinctes, j'ai étudié la contribution des préférences de compagnon mâle à l'isolement sexuel entre deux espèces de mouches des fruits étroitement liées subissant des coûts différentiels d'hybridation, Drosophila recens et Drosophila subquinaria. Les préférences des mâles sont particulièrement intéressantes en raison de leurs contributions potentielles à l'isolement sexuel, une forme d'isolement reproductif qui peut contribuer à la spéciation des espèces se reproduisant sexuellement. Dans la première expérience, je teste la présence de préférences de compagnon mâle dans chacune des deux espèces et si la force relative de la préférence est concordante avec le coût de l'hybridation. J'ai constaté que les mâles de D. subquinaria courtisaient sans discernement à la fois leurs propres femelles (c'est-à-dire homospécifiques) et les femelles hétérospécifiques de D. recens. Alors que D. recens de l'allopatrie a montré un modèle similaire, ceux de la sympatrie courtisaient leurs propres femelles plus que les femelles hétérospécifiques, indiquant un modèle de déplacement du caractère reproducteur. Dans la deuxième expérience, je teste le rôle de l'apprentissage dans le contexte de ces préférences de compagnon masculin dans D. recens, et si l'apprentissage a également montré un modèle de caractéristique de reproduction. Je n'ai pas trouvé de preuve d'apprentissage dans la mesure où les mâles D. recens ne réduisaient pas leur intensité de fréquentation envers les femelles hétérospécifiques après avoir été rejetés par des femelles similaires. Par conséquent, je n'ai pas trouvé d'indication de déplacement du caractère reproducteur. Enfin, en ce qui concerne la divergence phénotypique post-copulatoire, j'ai étudié les différences dans l'expression des protéines du liquide séminal entre les populations expérimentales de D. melanogaster connaissant l'un des trois environnements d'accouplement, permettant différentes possibilités de compétition de compagnon et l'environnement dans lequel elle a eu lieu. Ces trois environnements d'accouplement incluent un environnement dans lequel la compétition entre partenaires était absente (MCabsent,), un dans lequel la compétition entre partenaires se produisait dans un petit environnement structurellement simple (MCsimple) et un dans lequel la compétition entre partenaires se produisait dans un environnement plus grand et un peu plus complexe (MCcomplexe,). Les fluides séminaux mâles sont particulièrement intéressants en raison de leur capacité à négocier la compétition post-copulatoire entre les mâles et, par conséquent, peuvent être utilisés pour manipuler les femelles dans l'intérêt de la forme physique d'un mâle, potentiellement à ses dépens (c'est-à-dire conflit sexuel). J'ai étudié la divergence dans une protéine du liquide séminal particulière impliquée dans un conflit sexuel, le peptide sexuel (Acp70A). Alors que les niveaux d'expression génique chez les mâles du traitement à trois accouplements ne différaient pas en moyenne, les quantités relatives stockées le faisaient, les mâles MCcomplexe portant significativement moins de peptide sexuel que les mâles MCabsent ou MCsimple (qui ne différaient pas les uns des autres). Ce résultat suggère que la compétition de partenaire et l'environnement dans lequel elle se produit jouent un rôle important dans la divergence des phénotypes des peptides sexuels.