Given multiple matings from females, sexual selection continues after insemination in the form of postcopulatory sexual selection. This process is formed by two main mechanisms: sperm competition (competition of sperm of two or more males for the fertilization of the eggs of the same female) and cryptic female choice. These act as powerful selective pressures for the evolution of reproductive biology of both males and females. The aim of this thesis is to analyze the relative importance of sperm competition and cryptic female choice in determining a male reproductive success. The study species is the guppy (Poecilia reticulata), a freshwater fish, ovoviviparous with internal fertilization. Using artificial insemination, I studied if females can obtain fecundity benefits from mating with colourful males (as predicted by Sheldon's Phenotype-linked fertility hypothesis), the role of sperm number and sperm quality for sperm competition success, repeatability of a male's fertilization success and the role of MHC genes in non directional cryptic female choice. This technique allows to control for many potential confounding variables, as order of mating, number of sperm inseminated and cryptic female choice. From the experiments, it emerges that directional processes in this species are more important for fertilization success rather than non directional processes, even if a part of variance in fertilization success is explained by a male's similarity for MHC genotype with the female.

Post-mating reproductive isolating mechanisms may be among the earliest reproductive barriers to emerge among incipient species. Trinidadian guppy, Poecilia reticulata, populations in the Caroni and Oropouche drainages in Northern Trinidad exhibit marked genetic divergence and provide an ideal system in which to search for these barriers. We inseminated virgin females with equal amounts of sperm from two males, a ‘native’ male from the female's own population and a ‘foreign’ male from the other drainage. Artificial insemination ensured that mating order and mate choice did not affect the outcome. Paternities were assigned to the resulting broods using microsatellite markers. As predicted, sperm from native males had precedence over foreign sperm. Moreover, this effect was symmetrical for both drainages. In contrast, we detected no native sperm precedence in controls, in which females received sperm from the same and another population within the same drainage. Our results show that gametic isolation can arise between geographically proximate, though genetically divergent, populations of a single species and highlight the potential role of this process in speciation.
Keywords: speciation, sperm competition, internal fertilization, genetic divergence, post-mating pre-zygotic isolation

Among different teleost fish species, diverse sex-determining mechanisms exist, including environmental and genetic sex determination, yet chromosomal sex determination with male heterogamety (XY) prevails. Different pairs of autosomes have evolved as sex chromosomes among species in the same genus without evidence for a master sex-determining locus being identical. Models for evolution of Y chromosomes predict that male-advantageous genes become linked to a sex-determining locus and suppressed recombination ensures their co-inheritance. In the guppy, Poecilia reticulata, a set of genes responsible for adult male ornaments are linked to the sex-determining locus on the incipient Y chromosome. We have identified >60 sex-linked molecular markers to generate a detailed map for the sex linkage group of the guppy and compared it with the syntenic autosome 12 of medaka. We mapped the sex-determining locus to the distal end of the sex chromosome. We report a sex-biased distribution of recombination events in female and male meiosis on sex chromosomes. In one mapping cross, we observed sex ratio and male phenotype deviations and propose an atypical mode of genetic sex inheritance as its basis.

Background
The guppy, Poecilia reticulata, is a well-known model organism for studying inheritance and variation of male ornamental traits as well as adaptation to different river habitats. However, genomic resources for studying this important model were not previously widely available.
Results
With the aim of generating molecular markers for genetic mapping of the guppy, cDNA libraries were constructed from embryos and different adult organs to generate expressed sequence tags (ESTs). About 18,000 ESTs were annotated according to BLASTN and BLASTX results and the sequence information from the 3' UTRs was exploited to generate PCR primers for re-sequencing of genomic DNA from different wild type strains. By comparison of EST-linked genomic sequences from at least four different ecotypes, about 1,700 polymorphisms were identified, representing about 400 distinct genes. Two interconnected MySQL databases were built to organize the ESTs and markers, respectively. A robust phylogeny of the guppy was reconstructed, based on 10 different nuclear genes.
Conclusion
Our EST and marker databases provide useful tools for genetic mapping and phylogenetic studies of the guppy.

Background
The diversity of visual systems in fish has long been of interest for evolutionary biologists and neurophysiologists, and has recently begun to attract the attention of molecular evolutionary geneticists. Several recent studies on the copy number and genomic organization of visual pigment proteins, the opsins, have revealed an increased opsin diversity in fish relative to most vertebrates, brought about through recent instances of opsin duplication and divergence. However, for the subfamily of opsin genes that mediate vision at the long-wavelength end of the spectrum, the LWS opsins, it appears that most fishes possess only one or two loci, a value comparable to most other vertebrates. Here, we characterize the LWS opsins from cDNA of an individual guppy, Poecilia reticulata, a fish that is known exhibit variation in its long-wavelength sensitive visual system, mate preferences and colour patterns.
Results
We identified six LWS opsins expressed within a single individual. Phylogenetic analysis revealed that these opsins descend from duplication events both pre-dating and following the divergence of the guppy lineage from that of the bluefin killifish, Lucania goodei, the closest species for which comparable data exists. Numerous amino acid substitutions exist among these different LWS opsins, many at sites known to be important for visual pigment function, including spectral sensitivity and G-protein activation. Likelihood analyses using codon-based models of evolution reveal significant changes in selective constraint along two of the guppy LWS opsin lineages.
Conclusion
The guppy displays an unusually high number of LWS opsins compared to other fish, and to vertebrates in general. Observing both substitutions at functionally important sites and the persistence of lineages across species boundaries suggests that these opsins might have functionally different roles, especially with regard to G-protein activation. The reasons why are currently unknown, but may relate to aspects of the guppy's behavioural ecology, in which both male colour patterns and the female mate preferences for these colour patterns experience strong, highly variable selection pressures.

Background
Comparisons of functionally important changes at the molecular level in model systems have identified key adaptations driving isolation and speciation. In cichlids, for example, long wavelength-sensitive (LWS) opsins appear to play a role in mate choice and male color variation within and among species. To test the hypothesis that the evolution of elaborate coloration in male guppies (Poecilia reticulata) is also associated with opsin gene diversity, we sequenced long wavelength-sensitive (LWS) opsin genes in six species of the family Poeciliidae.
Results
Sequences of four LWS opsin genes were amplified from the guppy genome and from mRNA isolated from adult guppy eyes. Variation in expression was quantified using qPCR. Three of the four genes encode opsins predicted to be most sensitive to different wavelengths of light because they vary at key amino acid positions. This family of LWS opsin genes was produced by a diversity of duplication events. One, an intronless gene, was produced prior to the divergence of families Fundulidae and Poeciliidae. Between-gene PCR and DNA sequencing show that two of the guppy LWS opsins are linked in an inverted orientation. This inverted tandem duplication event occurred near the base of the poeciliid tree in the common ancestor of Poecilia and Xiphophorus. The fourth sequence has been uncovered only in the genus Poecilia. In the guppies surveyed here, this sequence is a hybrid, with the 5' end most similar to one of the tandem duplicates and the 3' end identical to the other.
Conclusion
Enhanced wavelength discrimination, a possible consequence of opsin gene duplication and divergence, might have been an evolutionary prerequisite for color-based sexual selection and have led to the extraordinary coloration now observed in male guppies and in many other poeciliids.

Well-studied model systems present ideal opportunities to understand the relative roles of contemporary selection versus historical processes in determining population differentiation and speciation. Although guppy populations in Trinidad have been a model for studies of evolutionary ecology and sexual selection for more than 50 years, this work has been conducted with little understanding of the phylogenetic history of this species. We used variation in nuclear (X-src) and mitochondrial DNA (mtDNA) sequences to examine the phylogeographic history of Poecilia reticulata Peters (the guppy) across its entire natural range, and to test whether patterns of morphological divergence are a consequence of parallel evolution. Phylogenetic, nested clade, population genetic, and demographic analyses were conducted to investigate patterns of genetic structure at several temporal scales and are discussed in relation to vicariant events, such as tectonic activity and glacial cycles, shaping northeast South American river drainages. The mtDNA phylogeny defined five major lineages, each associated with one or more river drainages, and analysis of molecular variance also detected geographic structuring among these river drainages in an evolutionarily conserved nuclear (X-src) locus. Nested clade and other demographic analyses suggest that the eastern Venezuela/ western Trinidad region is likely the center of origin of P. reticulata. Mantel tests show that the divergence of morphological characters, known to differentiate on a local scale in response to natural and sexual selection pressures, is not associated with mtDNA genetic distance; however, TreeScan analysis identified several significant associations of these characters with the haplotype tree. Parallel upstream/downstream patterns of morphological adaptation in response to selection pressures reported in P. reticulata within Trinidad rivers appears to persist across the natural range. Our results together with previous studies suggest that, although morphological variation in P. reticulata is primarily attributed to selection, phylogeographic history may also play a role.

Heat denaturation experiments revealed heat stability differences at a locus encoding glucosephosphate isomerase (GPI) in the guppy. Inheritance experiments indicated that the observed differences in heat stability are controlled by a single incomplete dominant autosomal locus.

Visual pigments initiate vision and are characterized by their wavelengths of maximal absorption (λmax). Modifications of the λmax values of visual pigments have allowed organisms to adapt to diverse light environments. The availability of the functional assays of these visual pigments using cultured cells makes vision an ideal genetic system to study the molecular bases of adaptation and genetics of color vision in vertebrates. The visual pigments in vertebrate retinas are distinguished into five evolutionarily distinct groups RH1 (λmax = 490-500 nm), RH2 (470-510 nm), SWS1 (360-420 nm), SWS2 (440-455), and LWS/MWS (510-570 nm). Here, we review amino acid replacements that are associated with the shifts in the λmax values of these visual pigments.
The λmax-shifts in several RH1 pigments seem to reflect adaptive changes to blue environments of the organisms and are explained mostly by amino acid replacements D83N (D → N at residue 83), E122Q, and A292S. Similarly, the blue-shifts in the λmax values of the RH2 pigments can be explained by D83N, E122Q, A164S, and M207L. For the SWS1 pigments of birds, only one amino acid replacement S84C seems to be responsible for the transformation of ultraviolet pigments from the violet pigment. For the LWS/MWS pigments, the additive effects of amino acid differences at 180, 197, 277, 285, and 308 fully explain the red-green color vision in a wide range of vertebrates. All of these observations suggest that the evolution of the extant visual pigments can be explained by amino acid replacements at only a small number of sites.
Visual pigments, a group of G-protein-coupled receptors, initiate visual excitation (Wald 1968). Each visual pigment consists of a transmembrane protein, opsin, and the chromophore, 11-cis-retinal, and can be characterized by its wavelength of maximal absorption (λmax). Human color vision is mediated by `blue', `green', and `red' visual pigments. The `blue' pigments absorb wavelengths ranging from about 370 nm to 570 nm with a λmax at 420 nm, while both `green' and `red' pigments are sensitive to wavelength about 450-620 nm with λmax values at 530 nm and 560 nm, respectively (Nathans 1989). The molecular bases of the spectral tuning of these and other visual pigments in vertebrates are still not well understood. Recently, however, some significant progress has been made on this subject.
To evaluate the mechanisms of the functional properties of visual pigments, a large number of amino acid changes have been introduced into the bovine rod-specific visual pigment (rhodopsin) by several groups of vision scientists (for a review, see Yokoyama 1997). In most of these analyses, charged amino acids have been considered. For example, an amino acid change from glutamic acid at residue 113 (E113) to glutamine (E113Q) shifts the λmax of the pigment from 500 nm to 380 nm (Sakmar et al. 1989; Zhukovsky and Oprian 1989; Nathans 1990a, b). E113 is the negatively charged counterion to the positively charged protonated Schiff base and the modification of this opsin structure causes a drastic shift in the λmax value of the pigment. Unfortunately, most of these amino acid changes, including E113Q, have not been found in nature. Thus, it is not immediately clear how these mutagenesis results are helpful in elucidating the molecular basis for the divergence of λmax values of visual pigments in nature (Yokoyama 1995, 1997).