Polyandry and postcopulatory sexual selection in yellow dung flies

Abstract

Sexual selection arises because individuals vary in reproductive success. It is dominated by two main processes: male competition for access to females (intrasexual selection) and mate choice exerted by choosy females (intersexual selection). Although these categories are most often envisioned to occur before mating, sexual selection is not limited to precopulatory processes, and securing mates is often insufficient for acquiring reproductive success. The two categories of sexual selection also occur after mating, predominantly via two postcopulatory mechanisms: male ejaculates compete for fertilization (sperm competition) and females may exert a preference for the sperm of certain males (cryptic female choice). Some aspects of postcopulatory sexual selection remain controversial, partly because many of these processes are hidden within the bodies of females, and therefore processes determining fertilization success are often inferred from patterns of paternity. As a consequence, mechanisms underlying sperm storage and utilization are largely unknown, and data that directly link the number of stored sperm to paternity are extremely scarce. Hence, the relative contributions of male (sperm competition) and female (cryptic female choice) mechanisms to differential fertilization success, and the extent to which these forces interact, are currently unknown. Polyandry (the mating of females with more than one male) is a prerequisite for postcopulatory sexual selection. It is a very common phenomenon in insects, but the evolutionary forces favouring multiple mating by females remain very controversial. This is especially true if there are no obvious direct benefits associated with female remating, for example the replenishing of females’ sperm stores or the acquisition of nutrients from mating partners. In such cases, repeated mating by females might arise via a number of alternative nonadaptive or adaptive mechanisms, including the acquisition of high quality or compatible genes (indirect benefits). The relative importance of each of these alternatives is currently unknown both in general and for many specific examples of female polyandry. This is partly because research on polyandry has relied heavily on laboratory experiments and it is often unclear whether laboratory findings reflect the conditions experienced by wild populations. Levels of polyandry observed in the lab are particularly suspect because the high densities of lab cultures may encourage more intersexual encounters and harassment than are present in the field. Therefore, to advance our understanding of the evolutionary causes and consequences of polyandry, we need more information on natural levels of polyandry in wild populations, ideally featuring analyses of its spatial and/or temporal variation. The yellow dung fly, Scathophaga stercoraria, is a model system for studying sexual selection and postcopulatory sexual selection in particular. Even so, field data on sperm storage and paternity are scarce, and the precise mechanisms underlying non-random paternity still unknown. The goal of my PhD thesis was to develop a microsatellite competitive PCR method for quantifying relative contributions of different males to sperm in storage, and apply this method to investigate sperm transfer, storage and utilization (e.g. to establish the relationship between the number of stored sperm and achieved paternity success). The second important purpose was to collect good field data on sperm storage and paternity. Genotyping the sperm stores of females additionally provided a useful estimate of prevailing levels of polyandry in a natural population of dung flies. In addition to direct studies of the mechanisms involved in sperm transfer, storage and utilization, I was also interested in tests of models of female preference evolution in a quantitative genetic framework, especially insofar as they might shed light on postcopulatory sexual selection mechanisms. Chapter 1 is an essay about a sexually selected sperm process in the dung beetle Onthophagus taurus initially described by Leigh Simmons and Janne Kotiaho. The “sexually selected sperm hypothesis” proposes that postcopulatory sexual selection selects for male traits that increase fertilization efficiency and female traits that promote sperm competition (e.g. multiple mating, complex female reproductive tracts). This hypothesis includes the sexy sperm mechanisms (enhanced fertilization success without enhancement of other fitness-related traits), but does not exclude the possibility that overall genetically superior males have greater fertilization efficiency (the good sperm mechanism). Simmons and Kotiaho applied a quantitative genetic approach to the dung beetle Onthophagus taurus to test this idea. They found significant additive genetic variation in spermatheca size, a trait that could play a central role in determining paternity biases. Importantly, consistent with sexy sperm and good sperm processes, their study shows that there is a significant negative genetic correlation between spermatheca size and sperm length: fathers that sired sons with short sperm also sired daughters with large spermathecae. These results acquire further significance when placed in the context of previous findings in Onthophagus. Shorter sperm have a fertilization advantage in competitive situations, and this advantage depends on spermatheca size. Sperm length, like spermatheca size, exhibits significant additive genetic variance and, interestingly, males in better condition produce shorter sperm. As a result of the genetic covariance between sperm length and male condition, females fertilizing their eggs using shorter sperm could produce offspring of high condition (the good sperm mechanism). Taken together, these findings suggest a sexually selected sperm process incorporating a (good sperm) mechanism to produce high-quality offspring. Postcopulatory sexual selection could thus shape sperm just like precopulatory female preferences affect evolutionary divergence of male secondary sexual traits. This chapter therefore introduces the compellingly complex interactions underlying postcopulatory sexual selection. The remaining chapters represent empirical efforts to disentangle this complexity using molecular studies of sperm storage and use in the lab and the field. One methodological challenge in the study of postcopulatory sexual selection is to quantify sperm transfer and storage of individual ejaculates within the reproductive tract of multiply mated females. Previously applied techniques such as radiolabelling and phenotypic markers are practically inferior to genetic markers, because they suffer from potentially confounding influence on sperm movement or completely unambiguous assignment is impossible. Chapter 2 describes the development and application of microsatellite competitive PCR for quantifying relative contributions to a small number of sperm in storage. We studied how DNA template characteristics affect PCR amplification of known concentrations of mixed DNA, and generated regressions for correcting observations of allelic signal strength based on such characteristics. We used these methods to examine patterns of sperm storage in twice- mated female yellow dung flies, Scathophaga stercoraria. We confirmed previous findings supporting sperm displacement and demonstrated that average paternity for the last mate accords with the mean proportion of sperm stored. We further found consistent skew in storage across the three sperm storage organs (spermathecae), with more last male sperm stored in the singlet spermatheca than in either doublet. We also showed that the time between copulations may be important for effectively sorting sperm. Finally, we demonstrated that male size may influence the opportunity for sperm choice, suggesting future work to disentangle the roles of male competition and cryptic female choice. Using the competitive PCR method developed in the previous chapter, chapter 3 assessed how biases in sperm storage relate to sperm use during oviposition and female reproductive anatomy. Importantly, by genotyping all offspring from potentially mixed-paternity clutches we directly estimated the relationship between stored sperm (S2) and paternity success (P2) of the second male. According with the previous chapter, we found consistent skew in sperm storage across the three spermathecae, with more second male sperm stored in the singlet spermatheca than in the doublet. S2 values generally decreased with increasing spermatheca size, possibly indicating less efficient sperm displacement in large spermathecae. Additionally, copula duration and several two-way interactions that included spermathecal identity, female size, and size of the second male significantly influenced S2, highlighting the complexity of postcopulatory processes and sperm storage. Mean S2 for the flies for which we genotyped all offspring was 59.8 % and matched P2 in those flies which was 58.7 %. Importantly, P2 and individual spermathecal S2 values were strongly correlated: 0.902 for the singlet spermatheca; 0.863 for the middle doublet spermatheca; and 0.836 for the outer doublet spermatheca. Oviposition-treatment strongly influenced S2, with S2 being smallest when females laid their eggs directly after the second copula. We argued that the act of laying eggs itself interrupted continued sperm transfer and displacement and caused the smaller S2 values. Interestingly, and contrary to prediction, S2 values were higher when females did not lay eggs than when they oviposited between copulations. Additional analyses across oviposition treatments indicated that reduced copula duration with post-oviposition females (e.g. strategic sperm allocation) explained this pattern. Our study therefore supports the complex network of factors suspected to influence sperm storage. The strong link between the proportion of stored sperm and paternity is most parsimoniously explained by sperm usage that is largely proportional to sperm storage. Nevertheless, substantial unexplained variance and the apparent bias across spermathecae in usage during fertilization could reflect a certain degree of sperm selection by females. Many more data such as these will help clarify the relative contributions of male (sperm competition) and female (cryptic female choice) mechanisms to differential fertilization success. Polyandry is a prerequisite for postcopulatory sexual selection and very common in insects. Nevertheless, the evolutionary causes and far-reaching consequences of this phenomenon remain debated. Chapter 4 presents a study of temporal variation in sperm storage and levels of polyandry in a natural population of yellow dung flies. We captured wild female yellow dung flies over the whole spring season and genotyped the sperm from their spermathecae to obtain field information on sperm transfer, storage, and associated levels of polyandry. On average females stored sperm from 2.47 males based on a minimum estimate, and 3.33 based on a probabilistic estimate that incorporates population allele frequencies, respectively. Sperm storage and therefore sperm competition intensity showed high temporal variation: the proportion of multiply mated females (i.e. females with sperm from ≥ 2 males within their sperm stores) and the absolute number of ejaculates detected within females strongly increased over the spring season before it sharply decreased at the end. Future studies should investigate how males respond to this varying competitive situation. Interestingly, we detected a positive relationship between the number of stored ejaculates and females’ wing injuries, suggesting a mechanism by which males may be able to assess prevalent levels of sperm competition intensity. In addition, the number of ejaculates differed amongst the three spermathecae. In agreement with the two previous chapters that documented highest sperm displacement in the singlet spermatheca following double matings in the laboratory, we detected fewer ejaculates in the singlet spermatheca than in either doublet. Currently, we cannot determine whether this skew across spermathecae of wild flies is adaptive, however, some kind of storage bias is a prerequisite for adaptive sperm selection. These field data on sperm transfer and storage provide an important extension to controlled laboratory experiments, and they are essential to validate empirical assessments of the causes and implications of polyandry in laboratory settings. Chapter 5 was also a field project and built on the previous chapter. I performed an oviposition site choice experiment in a natural population in which female yellow dung flies S. stercoraria could deposit their eggs into three different micro-environments on a dung pat (the ridge, north- or south-exposed side), and genotyped the offspring and sperm remaining after oviposition from the three spermathecae of these flies. Temperature strongly influenced egg placement: the warmer the temperature, the higher the proportion of eggs laid into the north-exposed side of dung. The number of ejaculates in storage differed amongst spermathecae as in the previous chapter and females stored sperm from more males than fathered their offspring. Mean last male paternity was 83.4 %, roughly matching some previous estimates from the laboratory, but higher than the reported 58.7 % in chapter 3. Importantly, I found no indication that females are able to lay eggs of different genotypes, by biasing paternity towards certain males, in different places. Thus this represents a strong test of adaptive sperm selection that failed to find any supporting evidence of it. However, this experiment did reveal positive effects of multiple mating on the total number and proportion of offspring emerging from dung. More studies that directly investigate polyandry and cryptic female choice in natural populations will be crucial for critically testing the importance of sperm selection relative to other aspects of postcopulatory sexual selection. Sexuelle Selektion entsteht weil sich Individuen in ihrem Reproduktionserfolg unterscheiden. Zwei Prozesse dominieren dabei: Männchenkonkurrenz um Zugang zu den Weibchen zu erhalten (intrasexuelle Selektion) und Partnerwahl ausgeübt durch wählerische Weibchen (intersexuelle Selektion). Einen Partner zu ergattern reicht allerdings nicht aus um den Reproduktionserfolg sicherzustellen. Daher finden die zwei Hauptprozesse nicht nur vor der Paarung statt (prekopulatorische sexuelle Selektion), sondern auch während und nach der Paarung (postkopulatorische sexuelle Selektion). Analog zu den zwei Prozessen vor der Paarung, dominieren zwei postkopulatorische Mechanismen: Die Ejakulate der Männchen konkurrieren um die Befruchtung der Eier (Spermienkonkurrenz) und die Weibchen können Spermien gewisser Männchen bevorzugen (kryptische Weibchenwahl). Gewisse Aspekte der postkopulatorischen sexuellen Selektion sind umstritten. Dies liegt zum Teil daran, dass diese Art der Selektion versteckt in den Weibchen drinnen stattfindet, und deswegen die Prozesse, die den Fortpflanzungserfolg bestimmen, häufig nur von der erzielten Vaterschaft abgeleitet werden. Daher sind die Mechanismen, die der Speicherung und Verwendung von Spermien zugrunde liegen weitgehend unbekannt. Des Weiteren sind Daten, welche die gespeicherte Menge der Spermien direkt mit dem erzielten Vaterschaftserfolg in Beziehung setzten, extrem selten. Infolgedessen ist zum jetzigen Zeitpunkt der relative Einfluss der männlichen (Spermienkonkurrenz) und weiblichen (kryptische Weibchenwahl) Mechanismen, sowie deren Interaktion, auf den unterschiedlichen Fortpflanzungserfolg der Individuen, unbekannt. Polyandrie (die Paarung von Weibchen mit mehr als einem Männchen) ist eine Voraussetzung für die postkopulatorische sexuelle Selektion. Obwohl das Phänomen unter Insekten weit verbreitet ist, sind die evolutionären Kräfte, die mehrfache Paarungen der Weibchen begünstigen, umstritten. Dies trifft vor allem dann zu, wenn Weibchen keinen offensichtlichen direkten Nutzen aus mehrfachen Paarungen ziehen, wie zum Beispiel das Nachfüllen ihres Sperma Vorrates oder den Erhalt von Nahrung vom Paarungspartner. In solchen Fällen kann mehrfaches Verpaaren der Weibchen durch eine Reihe von nicht- adaptiven oder adaptiven Mechanismen, wie zum Beispiel die Akquisition von guten oder kompatiblen Genen (indirekter Nutzen), entstehen. Die relative Bedeutung dieser alternativen Mechanismen für die Entstehung und Erhaltung von Polyandrie im Allgemeinen, aber auch für zahlreiche spezifische Beispiele wo sich Weibchen mehrfach paaren, ist zurzeit unbekannt. Ein wichtiger Grund dafür ist die Tatsache, dass Forschung über Polyandrie vor allem im Labor stattfindet, und wir deswegen oft nicht wissen, ob unsere experimentellen Designs eine realistische (natürliche) Situation darstellen. Laborresultate können deswegen auch nicht einfach eins zu eins ins Feld übertragen werden. Um unser Verständnis der evolutionären Ursachen und Folgen von Polyandrie zu verbessern, brauchen wir folglich mehr und bessere Daten bezüglich des Levels (Ausmass und Höhe) von Polyandrie in natürlichen Populationen. Idealerweise sollten Studien auch deren räumliche und zeitliche Variation untersuchen. Die Gelbe Dungfliege, Scathophaga stercoraria, ist ein Modellorganismus um die sexuelle Selektion und vor allem die postkopulatorische sexuelle Selektion zu studieren. Nichtsdestotrotz sind Felddaten betreffend Spermien Speicherung und Vaterschaft Mangelware, und die genauen Mechanismen, die der nicht-zufälligen Vaterschaft zugrunde liegen, noch immer unbekannt. Das Ziel meiner Doktorarbeit war es, eine kompetitive Mikrosatelliten PCR zu entwickeln, mit welcher man kleinste Spermien Mengen verschiedener Männchen im Fortpflanzungsapparat der Weibchen quantifizieren kann, und damit die Übertragung, Speicherung und Verwendung von Spermien (z.B. Beziehung zwischen gespeicherter Spermienmenge und erzieltem Vaterschaftserfolg) zu untersuchen. Das zweite wichtige Unterfangen meiner Dissertation bestand darin, wichtige Felddaten bezüglich Spermien Speicherung und Vaterschaft in natürlichen Populationen zu erhalten. Durch das Genotypisieren der Spermien in den Spermien-Speicherorganen der Weibchen (Spermatheken; die Gelbe Dungfliege hat drei: eine einzelne Spermatheke und eine „Doppelspermatheke“) erhält man gleichzeitig auch einen wertvollen Schätzwert über das Level von Polyandrie in natürlichen Populationen von Gelben Dungfliegen. Zusätzlich zu meinen Projekten, die direkt die involvierten Mechanismen bei der Spermien Übertragung, Speicherung und Verwendung untersucht haben, war ich auch an quantitativen genetischen Studien interessiert, welche Modelle der Evolution von Weibchen-Präferenzen getestet haben. Ich interessiere mich für solche Studien, weil diese ein grosses Potential haben Mechanismen der postkopulatorischen sexuellen Selektion zu beleuchten. Kapitel 1 ist ein Essay über einen sexuell selektierten Spermien-Prozess im Dungkäfer Onthophagus taurus, der ursprünglich von Leigh Simmons und Janne Kotiaho beschrieben wurde. Die „sexuell selektierte Spermien-Hypothese“ schlägt vor, dass die postkopulatorische sexuelle Selektion Männchen Merkmale selektiert (auswählt, bevorzugt), die die Befruchtungs-Effizienz erhöhen, und gleichzeitig aber auch Weibchen Merkmale selektiert, die die Spermienkonkurrenz fördern (z.B. Mehrfach-Paarungen, komplexe weibliche Fortpflanzungsapparate). Die Hypothese beinhaltet einen „sexy sperm“ Mechanismus (verbesserter Befruchtungserfolg, ohne dass andere Fitnesskomponenten verbessert sind), schliesst aber die Möglichkeit, dass allgemein genetisch bessere Männchen eine grössere Befruchtungs-Effizienz haben („good sperm“ Mechanismus) nicht aus. Simmons und Kotiaho haben diese Hypothese in einer quantitativen genetischen Studie mit Onthophagus taurus getestet. Sie entdeckten signifikante additive genetische Varianz für die Grösse der Spermatheken (Spermien-Speicherorgane der Weibchen). Wichtig, passend zu „sexy sperm“ und „good sperm“ Prozessen zeigte die Studie auch eine signifikante negative genetische Korrelation zwischen Spermathekengrösse und Spermienlänge auf: Väter, die Söhne mit kurzen Spermien zeugten, zeugten gleichzeitig auch Töchter mit grossen Spermatheken. Frühere Studien hatten bereits gezeigt, dass kurze Spermien einen Befruchtungsvorteil besitzen, dass die Spermienlänge signifikante additive genetische Varianz aufweist, und dass Männchen in besserer Kondition kürzere Spermien produzieren. Zusammengefasst legen diese Ergebnisse einen sexuell selektierten Spermien-Prozess nahe, der auch einen „good sperm“ Mechanismus beinhaltet um genetisch hochwertige Nachkommen zu produzieren. Dieses Kapitel führt in die fesselnde Komplexität postkopulatorischer sexueller Prozesse ein. Die nachfolgenden Kapitel versuchen empirisch, mittels molekularen DNA Methoden, die Komplexität der postkopulatorischen Mechanismen im Labor und Feld zu entschlüsseln. Eine besonders schwierige methodische Herausforderung beim Studium der postkopulatorischen sexuellen Selektion ist das Quantifizieren der übertragenen und gespeicherten Spermien verschiedener Männchen innerhalb des weiblichen Fortpflanzungsapparates. Bereits angewandte Techniken wie das Markieren durch Radioaktivität oder phänotypische Marker weisen Schwächen auf. Kapitel 2 beschreibt die Entwicklung und Anwendung einer kompetitiven Mikrosatelliten PCR mit der man kleinste Spermienmengen verschiedener Männchen in den Spermien-Speicherorganen der Weibchen quantifizieren kann. Wir studierten wie die Eigenschaften der DNA Matrize (template) die PCR Amplifikation von bekannten Konzentrationen von