Extent of Hybridization and Reproductive Isolation in Daphnia

Eawag_04786

Doctoral Thesis ETH No. 16685

EXTENT OF HYBRIDIZATION AND REPRODUCTIVE ISOLATION IN DAPHNIA

A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH

for the degree of Doctor of Sciences

presented by

BARBARA KELLER Dipl. Natw. ETH born on 16.01.1972 citizen of Märstetten (TG) and Konolfingen (BE)

accepted on the recommendation of

PD. Dr. Piet Spaak, examiner Prof. Dr. Nelson G. Hairston Jr., co-examiner Prof. Dr. Jukka Jokela, co-examiner

2006 für meinen Vater

TABLE OF CONTENTS

Summary 1

Zusammenfassung 3

Chapter 1: Introduction and outline of the thesis 7

Chapter 2: Ephippia and Daphnia abundances under

changing trophic conditions 25

Chapter 3: Non-random sexual reproduction and diapausing

egg production in a Daphnia hybrid species complex 35

Chapter 4: Pre- and postzygotic reproductive isolation keep

hybridizing Daphnia species distinct 55

Chapter 5: Anthropogenic impact on hybrid success and taxa

niche separation in Daphnia 73

Chapter 6: Concluding remarks and recommendations for further research 91

Curriculum vitae 107

Danksagung 113

SUMMARY

Hybridization and introgression are sources of genetic variation that can accelerate adaptation and speciation or slow them down. Hybridization occurs after secondary contact (i.e. two species meet after a period of temporal and/or spatial isolation) between partially reproductively isolated species. The evolutionary significance of hybridization, however, depends strongly on the frequency of hybridization events, on hybrid fitness and whether hybrid lineages can be maintained after their formation. Therefore, several potential outcomes of hybridization can be expected: it can lead to extinction of one or both of the two parental species, to coexistence of parental species and hybrids, to reinforcement (i.e. natural selection against the production of unfit hybrids), or to merging into novel populations of reticulate or polyphyletic origin due to introgressive hybridization. Hybridization might be especially important in species that can propagate both asexually and sexually, such as the cyclic parthenogenetic Daphnia. In Daphnia, long asexual phases are interrupted by short periods of environmentally induced sexual reproductive phases. Sexually produced eggs are released within a protective structure called an ephippium and hatch after a period of diapause when exposed to hatching stimuli. Ephippia are important for Daphnia to colonize new habitats, to re-colonize local populations after unfavourable events and to maintain or increase genetic variation. They can stay viable for decades and be found in so called diapausing egg banks in the sediment; equivalent to the seed banks of plants. The research described in this thesis focuses on the question of which endogenous and exogenous selective factors affect interspecific hybridization and the coexistence of taxa within the Daphnia galeata-hyalina-cucullata species complex. In Chapter 2, we compared the applicability of ephippia preserved in the sediment with the regular monitoring of the pelagic population (i.e., quantifying sexual females) to reconstruct the trophic history using Greifensee (Switzerland) as a model system. We found a good match of the past total phosphorus concentration with the abundance of ephippia in the sediment, but not with the regular monitoring data of the pelagic sexual females. We thus suggested that sexually produced ephippia are useful biological indicators to reconstruct the trophic history of lakes. In Chapter 3, we tested if hatchlings obtained from an ephippial egg bank can be used to reconstruct former Daphnia taxon assemblages. We compared the taxon composition of recently produced ex-ephippial hatchlings with the distribution of present asexual females and sexual stages (sexual females and males). Using

- 1 - Summary these data, we extrapolated to the egg bank (i.e. taxon composition of hatchlings from the sediment). We found a significant taxon shift between the present pelagic population (F1 hybrid dominated) and hatchlings (predominantly D. galeata), both the recently produced ones as well as those from the sediment. We hypothesized that this taxon shift may be based on reproductive isolation mechanisms between D. galeata and D. hyalina. Such a taxon shift between the pelagic population and ex-ephippial hatchlings was also found previously in Lake Constance. Hence, in Chapter 4, I focussed on the question whether prezygotic (e.g., assortative mating, temporal isolation) or postzygotic (e.g., hybrid inviability or infertility) isolation is more likely to affect the hybridization between the two taxa. We found evidence for reproductive isolation (mainly post-zygotic). Hybrids dominate the asexual Daphnia population even though they have reduced sexual fitness, providing one of the potential mechanisms for parental taxa to remain distinct, coexisting types. In Chapter 5, we analyzed the effect of environmental parameters on taxon composition with the main focus on trophic changes (i.e., anthropogenic pollution) among 43 lakes in Switzerland and Northern Italy. We found that lake size, altitude and total phosphorus concentration are sufficient to describe niche breadths between Daphnia taxa. Hybrid dominance, however, could only be explained when the magnitude of the change in phosphorus content (proxy for habitat disturbance and trophic history) was incorporated in the analysis. In summary, although the cyclic parthenogenetic life cycle of Daphnia may facilitate the outcome of hybridization (asexual propagation allows sexually less fit hybrids to reach high abundances), we propose that human activities have been of primary importance in mediating hybridization and in altering Daphnia taxon composition. Thus, habitat disturbance (over- fertilization/eutrophication of lakes in the last century and more recent restoration/oligotrophication) influenced not only ephippial abundance in the sediment but also had a lasting effect on recent Daphnia taxon composition. In Chapter 6, I conclude the thesis with a discussion how molecular markers, which are now available in larger numbers, may help to clarify some of the remaining questions about the complex hybridization history in Daphnia.

- 2 - ZUSAMMENFASSUNG

Zwischenartliche Hybridisation (Kreuzung zweier Arten) und Introgression (Einführung von Genen einer Art in den Genbestand einer anderen durch wiederholte Kreuzung und Rückkreuzung) gelten als Quellen für genetische Variabilität und können den Anpassungs- und Artbildungsprozess beschleu- nigen oder verlangsamen. Zwischenartliche Hybridisation findet zwischen zwei in ihrer Fortpflanzung teilweise getrennten Arten statt, welche sich vor- gängig zeitlich und/oder räumlich getrennt voneinander entwickelt haben. Ob Hybridisation die Evolution zu beeinflussen vermag, hängt von der Kreuzungs- häufigkeit, der Hybridfitness und der Stabilität bzw. Überlebensfähigkeit der hybriden Linien ab. Die folgenden Szenarien können aus der Kreuzung zweier Arten resultieren: Aussterben einer der zwei Elternarten, Zusammenleben der Elternarten mit den Hybriden, Verstärkung von Artbarrieren oder Verschmel- zung der zwei Arten zu einer neuen Population mit netzartigem beziehungs- weise polyphyletischem (von mehreren Ahnen abstammend) Ursprung. Zwischen Arten, wie zum Beispiel den zyklisch parthenogenetischen Wasserflöhen (Daphnia), welche sich sowohl sexuell als auch asexuell vermehren, kann Hybridisation besonders wirksam sein. Bei Wasserflöhen wechseln sich lange asexuelle Perioden mit kurzen, durch Umweltänderungen ausgelösten sexuellen Perioden ab. Sexuell produzierte Eier, so genannte Dauereier, werden dabei in eine schützende Hülle (Ephippium) entlassen. Dauereier können nachdem dem sie eine Ruhephase (Diapause) überdauert haben, durch entsprechende Anreize zum Schlüpfen gebracht werden. Ephippien sind für Daphnien wichtig, da sie die Besiedlung neuer oder die Wiederbesiedlung vorübergehend unbewohnbarer Lebensräume ermöglichen und die genetische Variabilität aufrechterhalten oder erhöhen helfen. Dauereier können jahrzehntelang lebensfähig bleiben und bilden einen Dauereiervorrat im Seesediment (entsprechend dem Samenvorrat der Pflanzen im Boden). Die Forschung, die ich in dieser Doktorarbeit beschreibe, konzentriert sich auf die Frage, welche exo- und endogenen selektiven Faktoren die Hybridisation und Koexistenz der Taxa des Daphnia galeata-hyalina-cucullata Artenkomplexes beeinflussen. In Kapitel 2 untersuchten wir die Eignung eines Dauereiervorrats im Seesediment und eine regelmässige Langzeit-Plankton- Probenahme (d.h. Quantifikation der sexuellen Wasserflohweibchen) zur Re- konstruktion der trophischen Geschichte. Für diese Untersuchung benutzten wir den Greifensee als Modellsystem. Wir fanden eine gute Übereinstimmung zwischen der Gesamtphosphorkonzentration und der Häufigkeitsverteilung der

- 3 - Zusammenfassung

Ephippien im Dauereiervorrat des Seesediments. Keine Übereinstimmung hingegen ergab sich zwischen der Gesamtphosphorkonzentration und dem regelmässigen Plankton-Monitoring der sexuellen Weibchen. Hieraus schlos- sen wir, dass sich sexuell produzierte Ephippien als Bioindikatoren zur Rekon- struktion der trophischen Vergangenheit eines Sees eignen. In Kapitel 3 untersuchten wir die Anwendbarkeit der aus dem Dauereiervorrat geschlüpften Daphnien zur Rekonstruktion der Artenzusammensetzung der ehemaligen Seepopulation. Wir verglichen die Artenzusammensetzung von geschlüpften Daphnien aus aktuell produzierten Dauereiern mit derjenigen der asexuellen Weibchen und den sexuellen Stadien (sexuelle Weibchen und Männchen). Die so gefundenen Ergebnisse projizierten wir auf den Dauereiervorrat (d.h. auf die geschlüpften Daphnien aus dem Sediment). Wir fanden einen signifikanten Wechsel in der Artenzusammensetzung zwischen der Seepopulation (Domi- nanz der F1 Hybriden) und den Daphnien, die sowohl aus den heute produzierten Dauereiern als auch aus dem Dauereiervorrat geschlüpft sind (hauptsächlich D. galeata). Wir vermuteten, dass sich dieser Unterschied auf Isolationsmechanismen, die während der Fortpflanzung zwischen D. galeata und D. hyalina wirken, gründet. Ein vergleichbarer Wechsel der Artenzusam- mensetzung zwischen der Seenpopulation und den aus Dauereiern geschlüpf- ten Daphnien gibt es auch im Bodensee. Aus diesem Grund konzentrierte ich mich in Kapitel 4 auf die Frage, ob die Hybridisation zwischen den zwei Arten eher durch Isolationsmechanismen, die vor der Zellverschmelzung stattfinden (z. B. auswählende Paarung, zeitliche Isolation der Arten), oder durch solche, die nach der Zellverschmelzung entstehen (z. B. reduzierte Überlebensfähig- keit oder Sterilität der Hybriden), beeinflusst ist. In der Tat fanden wir Hin- weise auf Isolationsmechanismen, und zwar hauptsächlich auf solche, die nach der Zellverschmelzung stattfinden. Hybriden dominieren zwar klar die See- population, haben aber gleichzeitig eine verminderte sexuelle Fitness, was eine Erklärung dafür sein könnte, weshalb die Elternarten noch als solche vor- kommen und mit den Hybriden zusammenleben können. In Kapitel 5 untersuchten wir in 43 schweizerischen und nord- italienischen Seen die Auswirkung unterschiedlicher Umweltparameter auf die Daphnia-Artenzusammensetzung. Insbesondere interessierte uns dabei der Einfluss der trophischen Geschichte (d.h. von Menschen verursachte Über- düngung). Die Seengrösse, die Meereshöhe und die heutige Gesamtphosphor- belastung reichten aus, um die Nischenbreite der Wasserfloh-Arten zu be- schreiben. Die Dominanz der Hybriden hingegen konnte erst erklären werden, als wir die Geschwindigkeit und Höhe der Änderung der Gesamtphosphor-

- 4 - Zusammenfassung belastung (welche wir als Mass für die Störung und trophische Geschichte benutzten) mit in die Analyse einbezogen. Wenn auch die zyklisch parthenogenetische Lebensweise der Daphnien die Hybridisation zu erleichtern vermag (eine hohe asexuelle Vermehrungsrate erlaubt den Hybriden ihre sexuelle Unzulänglichkeit auszugleichen und trotzdem hohe Dichten zu erreichen), denken wir, dass die Veränderung in der Daphnia-Artenzusammensetzung hauptsächlich auf die menschlichen Aktivi- täten zurückzuführen ist. Es scheint, dass die Störung des Lebensraumes durch die Seenüberdüngung während des letzten Jahrhunderts und die nachfolgende Restaurierung nicht nur die Ephippia-Dichte im Sediment, sondern auch die heutige Artenzusammensetzung im See beeinflussten. In Kapitel 6 schliesse ich die Doktorarbeit mit einer Diskussion darüber, wie die heute in grösserer Zahl verfügbaren molekularen Marker helfen könnten die noch offenen Fragen über die komplexe Hybridisationsgeschichte der Daphnien zu klären.

- 5 -

CHAPTER 1

Introduction and outline of the thesis

Hybridization and its evolutionary significance Interspecific hybridization and introgression (Box 1), was first observed in plants as an important factor in the process of speciation, but during the past few decades many hybrid animal species have been detected, demonstrating its importance in the animal kingdom (reviewed by Barton and Hewitt 1989; Arnold 1992; Dowling and Secor 1997). However, plant species seem to hybridize more frequently (at least 25% of all species are involved in hybridization) than animal species (at least 10%, reviewed by Mallet 2005). The evolutionary significance of hybridization depends strongly on the frequency of hybridization events, on hybrid fitness and the ability of new recombinant genotypes to outperform one of their parents in at least some habitats (reviewed by Arnold 1992). Hybridization may lead to increased genetic diversity, and can be the origin for genetically novel adaptation (reviewed by Rieseberg and Carney 1998). By recombination of genetic material, the hybrid genotypes generated may be able to occupy novel environments (Barton 2001; Lexer et al. 2003; Cozzolino et al. 2006 ) and initiate hybrid speciation (reviewed by Bullini 1985; Rieseberg 1997). Antagonistically the evolution of barriers to gene flow (i.e. reinforcement) might be strengthened, due to natural selection, against the production of unfit hybrids (reviewed by Dobzhansky 1940; Turelli et al. 2001). Although many doubts exist on the evolutionary relevance of reinforcement in nature (Marshall et al. 2002), evidence meeting the criteria for the evolution of reinforcement were found, for instance, in a Triturus vulgaris × T. montandoni hybrid zone (Babik et al. 2003).

- 7 - Introduction Chapter 1

Hybridization can lead to the extinction of species (reviewed by Rhymer and Simberloff 1996). This aspect of hybridization and introgression has been studied theoretically (Epifanio and Philipp 2000; Wolf et al. 2001; Ferdy and Austerlitz 2002) as well as in natural populations with serious consequences for rare species (reviewed by Levin et al. 1996). The threat to endangered rare species due to hybridization raises concerns for management and conservation (reviewed by Allendorf et al. 2001, and references therein). However, common native species can also be threatened by hybridizing invasive species, as a study on California cordgrass and introduced smooth cordgrass (Ayres et al. 2004) and a theoretical model (Hall et al. 2006) showed. The invasiveness of introduced species can often be observed after long time lags, or multiple introductions, or both. One explanation for this phenomenon might be that invasiveness can evolve after species have started to hybridize with each other (Ellstrand and Schierenbeck 2000). A complete breakdown of reproductive barriers due to hybridization might induce a merging of the species into a hybrid swarm (reviewed by Seehausen 2004). Nevertheless, hybrid zones exist where a long-term coexistence of hybrids with parental species was described (e.g., Britch et al. 2001). Even swarms of highly fit hybrids can coexist with parental species without merging or being swamped, if selection favours hybrid individuals but not lineages, because favourable epistatic gene combinations can not be inherited between generations (Barton 2001; Burke and Arnold 2001), or if high hybrid fitness is extrinsically determined and restricted to the habitat of the hybrid zone (Moore 1977; Arnold 1997; Rieseberg and Carney 1998), or both.

Box 1. Hybridization and types of selection

Natural hybridization: Crossings of genetically distinguishable groups or taxa/species (i.e. interspecific hybridization, e.g., Arnold 1997).

Introgression (introgressive hybridization): Exchange of genes between evolutionary lineages due to backcrossing (e.g., Arnold 1992).

Two selection types can be discriminated that affect hybrid fitness (Moore 1977; Burke and Arnold 2001): Endogenous (epistatic) selection - selection against hybrid genotypes regardless of the environment (i.e. meiotic irregularities, physiological/developmental abnormalities of hybrid with mixed ancestry - hybrid incompatibility). Exogenous selection - it refers to environmental-specific fitness differences

- 8 - Chapter 1 Introduction

Abiotic and biotic impacts on hybridization Successful hybridization in natural populations can only be observed after the following events: 1) species have come into secondary contact with each other, which is influenced by natural or human induced dispersal and disturbance; 2) viable hybrid offspring have been produced, which depends, for example, on reproductive isolation mechanisms; 3) hybrids have successfully established, which depends mainly on their competitive abilities and thus in a broader sense on habitat conditions as well as on their reproductive mode. Therefore hybrid fitness can be influenced by a variety of endogenous and exogenous (Box 1) selective processes.

Box 2. Proposed models explaining the maintenance / stability of hybrid zones

Tension zone model - dispersal dependent model that assumes stability maintained by an equilibrium between endogenous selection against hybrids and the continuous dispersal of parental forms into the hybrid zone. An environmentally independent model with selection against intermediate genotypes (i.e. heterozygotes or recombinats) (Key 1968; Barton and Hewitt 1985).

Bounded hybrid superiority model - assumes that the selection stabilizing the hybrid zone is caused by genotype-by-environment interactions (i.e., exogenous selection) and occurs because of environmental heterogeneity. Different taxa are then favoured in different parts of the environment. In the hybrid zone, hybrids are assumed to be more fit than their parental taxa, whereas outside the hybrid zone, their fitness is lower (Moore 1977)

Mosaic model - this model assumes that hybrids are inherently less fit than their parents and that they occur rather patchily within a heterogeneous distribution range of the parental species. As a consequence the relative fitness of hybrids depends on environmental conditions within each patch (Harrison 1985; Howard 1986; reviewed in Arnold 1997).

Temporal hybrid superiority model - environment dependent model, which assumes increased hybrid fitness during specific time periods (i.e., fluctuating environmental conditions). This model was developed to explain co-existence between parental taxa and hybrids (Spaak and Hoekstra 1995).

Secondary species contact - Contact zones (i.e., hybrid zones) between species occur naturally at boundaries of species distributions/ranges. There are two main models that explain the maintenance and stability of hybrids in natural populations (see Box 2); the tension zone model (Barton and Hewitt 1985; Barton and Hewitt 1989) and the bounded hybrid superiority model (Moore 1977; Moore and Koenig 1986, Box 2). Derivatives from these two models

- 9 - Introduction Chapter 1 that assume no narrow hybrid zones or clines, are the mosaic model (Harrison 1985; Howard 1986; reviewed by Arnold 1997) and the temporal hybrid superiority model, proposed by Spaak and Hoekstra (1995), that takes temporal fluctuations into consideration (Box 2). Wide geographic regions (i.e., ‘suture hybrid zones’) have been found where formerly isolated species come into secondary contact with each other and unusual accumulations of hybridization can be observed. Such hybrid zones were attributed to Pleistocene climatic oscillations and associated contractions and expansions of species ranges (reviewed by Hewitt 2001; Swenson 2005). Historically, the survival of species as well as the genetic diversity of populations have been influenced by dispersal abilities, the occurrence of dispersal barriers (e.g., mountain chains), and the presence of glacial refuges (reviewed by Hewitt 2001). The present distribution range of the Potentilla species complex (Eriksen and Topel 2006) and D. pulex species complex (Weider and Hobaek 2003), for instance, can be explained by glacial refuges (e.g., Beringea), migration routes and hybridization. Recently increased global human activity, however, has accidentally or intentionally bridged natural species borders and barriers, increasing the probability for hybridization of formerly isolated species (e.g., Rhymer and Simberloff 1996). Habitats into which immigrants arrive are normally colonized by resident populations that are generally well buffered against invaders, due to local adaptation and large population sizes (Boileau and Hebert 1991; De Meester et al. 2002). Moreover, novel habitats are usually ecologically different from the native habitats of the immigrant, which could reduce the immigrant’s fitness and the likelihood of successful mating and hybridization (Nosil et al. 2005). Disturbance of resident populations can lead to ill-adapted populations, which might result in niche shifts of existing populations, or facilitate invasion and subsequent hybridization (reviewed by Moore 1977; Levin 2004). All these facts consistently suggest that present population structures rely on recent as well as on historical processes.

Viable hybrid offspring - Successful mating between genetically distinct species is thwarted by various kinds of reproductive isolation mechanisms, which can occur before, or after fertilization, or both (Box 3). These mechanisms can be found in various numbers, combinations and strengths between the species involved (Stebbins 1950; Howard 1993; Abernathy 1994; Arnold 1997). A general relation between the divergence times of parental species and reproductive incompatibility has been found; hence incompatibility

- 10 - Chapter 1 Introduction varies among taxonomic groups (reviewed by Edmands 2002). Reports of putative isolation mechanisms are numerous, but to disentangle which ones and to what extent premating, or postmating incompatibilities, or both, affect hybridization is difficult to ascertain. For instance, in natural populations of hybridizing ground crickets it was impossible to evaluate if the detected deviations were the result of assortative mating or a reproductive barrier operating prior to egg hatching (Howard et al. 1993). Further, it has been shown that a tendency toward unidirectional mating exists, with the more rare species as the maternal species (reviewed by Wirtz 1999). However, differences in the directionality mode might alter between closely related species. This was shown for three hybridizing Rorippa species, with bidirectionality between one hybrid pair (R. amphibian and R. sylvestris) and unidirectionality between the other one (R. amphibian and R. palustris) (Bleeker and Hurka 2001).

Hybrid establishment - The interference of distinct parental genomes can produce positive and negative epistatic effects on the nuclear and cytonuclear level and results in new genotypes which vary in fitness (Burke et al. 1998; Rieseberg and Carney 1998). However, the final success of hybrids in a specific habitat depends on complex genotype-by-environment interactions (Arnold et al. 2001) as well as on stochastic effects related to extinction and colonization (Babik et al. 2003). In a reciprocal transplant long-term experiment, the survivorship and fitness of artificially produced progeny in two naturally hybridizing species (Ipomopsis aggregate and I. tenuituba) were studied (Campbell and Waser 2001). All four cross types (both parents, both reciprocal F1 hybrids) were found to be equally fit in number and weight of seeds, however, the survival of these crosses after five years varied significantly, with the directionality of cross and environment type as significant factors. Besides environment-by-genotype interactions, the final success of hybrids further depends on their reproductive strategies (i.e. stabilization of breeding behaviour of hybrid lineages), as extensively discussed for plants with their various kinds of reproductive modes (reviewed by Rieseberg 1997). Hybridization might be especially important among plant and animal species that can propagate both asexually and sexually. Based on a transplant experiment with rhizomes (i.e., parents, F1 and F2 hybrids) of the Louisiana iris species complex, Emms and Arnold (1997) concluded that clonal reproduction may ensure long-term survival of early-generation hybrid genotypes. The

- 11 - Introduction Chapter 1 authors of another study of the same species complex suggested that even hybrids with reduced sexual fertility can reach high abundance due to clonal growth, which might increase their chance for sexual reproduction (Burke et al. 2000). Consequently asexual reproduction can facilitate hybrid dominance and the production of later hybrid generations (e.g., F2 hybrids and backcrosses), self-fertilization, and finally, introgression into the parental population. A comparable reproductive strategy, with alteration of asexual and sexual reproduction (i.e. cyclical parthenogenesis) and common hybridization, can be found in cladocerans (e.g., Daphnia) and rotifers, both freshwater organisms (reviewed by Hebert 1985).

Box 3. Classification of reproductive isolating mechanisms* Form of reproductive isolation: Description: Premating, prezygotic barriers Temporal, allochronic reduced encounters of potential mates due to isolation different mating times Habitat, ecological isolation reduced encounters of potential mates due to different mating sites Immigrant inviability, reduced encounters of potential mates due to premating mortality of maladapted immigrants Sexual, behavioural, reduced mating due to divergent courtship signals, ethological isolation mate preferences Postmating, prezygotic barriers Mechanical isolation reduced transfer of sperm during mating due to poor genitalic compatibility Gametic incompatibility reduced fertilization of eggs by ill-suited sperm Postmating, postzygotic barriers Immigrant inviability, reduced production of offspring due to mortality of postmating mated, maladapted immigrants Zygotic mortality reduced survival of zygotes soon after fertilization Hybrid inviability (genetic) reduced survival of hybrid offspring independent of the environment Hybrid inviability (ecological) reduced survival of hybrid offspring where viability is environment dependent Sexual selection against reduced mating success of hybrid offspring hybrids Hybrid sterility reduced fertility of hybrid offspring F2 breakdown reduced survival or fertility of subsequent hybrid generations * after Nosil et al. (2005) and references therein.

- 12 - Chapter 1 Introduction

asexual reproduction sexual reproduction

growth & physiological maturation change

ephippia sexual asexual asexual female juvenile amictic female parthenogenesis mating male male development

hatching eggs female development diapause

(Photos: P. Spaak & C. Rellstab) Figure 1. Reproduction in Daphnia. The left cycle represents the asexual reproduction during which parthenogenetic eggs are produced. When conditions become unfavourable, females produce sexual eggs and males (right cycle). Sexual reproduction (right cycle) allows for hybridization and results in diapausing egg production sheltered by a so-called ephippium. Later on, females hatch from these ephippial eggs. Some Daphnia species are able to produce ephippial eggs asexually (not shown here since it is not relevant for the species investigated in this thesis).

The Daphnia model system Daphnia (Crustacea, Cladocera) are the major herbivores in many lakes and ponds. Consequently, they are very important in aquatic food webs (e.g., Edmondson and Litt 1982; Carpenter et al. 1987). Their superiority as filter feeders is based on their ability to cope with various ranges of food types and sizes and on their extraordinary reproduction ability. Most Daphnia species are cyclical parthenogens, where sex is environmentally and genetically determined (e.g., Korpelainen 1989). Under favourable environmental conditions their reproduction rate is optimised by amictic parthenogenesis, where females produce clones of their own (Frey 1982). When conditions deteriorate Daphnia switch to sexual reproduction and produce males and sexual haploid eggs (ephippial eggs; Figure 1). In cyclically parthenogenetic Daphnia, ephippial eggs need to be fertilized (Hebert 1988), which might result in interspecific hybridization. A protective envelope, termed an ephippium, covers the two eggs. Through ephippial production Daphnia is able to

- 13 - Introduction Chapter 1 colonize new habitats and to re-colonize local populations after periods of unfavourable conditions. The protection that the ephippium provides allows ephippial eggs to survive gut passages (Mellors 1975) and survive freezing or desiccation (Innes and Singleton 2000; Bilton et al. 2001). The durability of ephippia enables their accumulation in the sediment where they build up a so called egg bank (e.g., De Stasio 1989).

dispersal from dispersal out other systems floating of system hatching diapausing egg ‘active‘ stimuli sediment production egg bank (especially accumu- (in litoral) light) hatching lation no hatching sedimentation ‘inactive‘ stimuli egg bank (in deep water)

Figure 2. Schematic visualization of a Daphnia egg bank in large lakes; ‘active’ (grey) and ‘inactive’ (black) egg bank (modified after Cáceres and Hairston 1998). The ‘active’ egg bank is characterized by sufficient presence of hatching stimuli. In contrast to shallow water bodies, hatching stimuli are restricted to the litoral zone, due to limited light penetration in deep water.

A special feature of the genus Daphnia is the ability of closely related

‘species’ to mate with each other, generating fertile F1 hybrids as well as fertile backcrosses (Gießler 1997). Hence morphologically closely related, coexisting ‘species’ are often a part of so-called species complexes. Therefore, parental Daphnia species and their hybrids do not form interpopulational transition zones (hybrid zones), but rather patchy spacial distributions of hybrids and parental species (Schwenk and Spaak 1997). Various species complexes in the genus Daphnia have been described (Hebert 1985; Wolf and Mort 1986; Taylor and Hebert 1992; Schwenk and Spaak 1997). The Daphnia longispina complex, for instance, contains eight species: hybrids between three of them, D. galeata, D. cucullata and D. hyalina (= D. galeata-hyalina-cucullata species complex) are commonly found sympatrically with their parental species in permanent lakes all across Europe (Spaak 1996; Schwenk and Spaak 1997). This co-occurrence of taxa can be explained by taxon-specific fitness differences indicated by specific niche breadths (Weider 1993). It has been shown that hybrids, under particular environmental conditions, can have a

- 14 - Chapter 1 Introduction higher fitness than their parental species (Schwenk and Spaak 1997). Hybrids may even become extremely abundant and sometimes almost out-compete one or both parental species (e.g., Schwenk and Spaak 1995). This phenomenon is explained by the temporal hybrid superiority model (Spaak and Hoekstra 1995, Box 2). However, long-term co-existence of parental species and hybrids (e.g., Spaak 1996) is possible either under conditions that also favour parental taxa and strengthen their competitive ability, or by the presence of reproductive isolation mechanisms that prevent parental species and hybrids from merging. For example, parasitism was found as possible factor strengthening the competitive ability of the parental taxon (Wolinska et al. 2004): D. galeata can be more resistant to a harmful parasite than its F1 hybrid offspring. Evidence for the presence of reproductive isolation mechanisms was derived from the observation that the taxonomic compositions between hatchlings originating from egg banks and active (asexual) lake populations differ significantly (e.g., Jankowski and Straile 2003). An egg bank consists of an ‘active’ and ‘inactive’ compartment (Cáceres and Hairston 1998, Figure 2). In the ‘active’ part ephippial eggs are capable of hatching and recolonize the pelagic population. Although the hatching process is not fully understood, light and elevated temperature may play a role in breaking dormancy (Schwartz and Hebert 1987). Because of thick sediment coverage, no hatching stimuli can reach the eggs in the ‘inactive’ egg bank. In contrast to egg banks of small and shallow water bodies (Cáceres and Hairston 1998), the ‘active’ egg bank of deep large lakes is restricted to the litoral zone, because of the absence of light in the deeper part of the lake (Figure 2). Several authors have shown that Daphnia egg banks can serve as archives to reconstruct microevolutionary changes over time (Weider et al. 1997; Hairston et al. 1999a; Hairston et al. 1999b; Cousyn et al. 2001; Limburg and Weider 2002) and that hatchlings can be used for experimental studies (resurrection ecology, e.g., Kerfoot and Weider 2004). For the reconstruction of former taxonomic compositions, however, one has to assume that the sexual eggs represent the pelagic asexual population. This assumption may be violated by elements of interference, which can occur before (e.g., pelagic asexual population reproduce assortatively) or after diapausing egg (e.g., taxon- specific survival or hatching success) production.

- 15 - Introduction Chapter 1

Box 4: Available diagnostic molecular markers in the D. galeata-hyalina-cucullata species complex

Allozymes - traditional diagnostic markers to discriminate between the three species and their hybrids. AO and AAT are species specific markers (Moore and Koenig 1986; Wolf and Mort 1986; Wolf 1987; Gießler 1997). Advantage: fast and relatively cheap. Disad- vantage: certain amount of fresh tissue is needed and hence, not applicable to diapausing eggs or ethanol preserved samples (therefore, only the hatchable part of an egg bank is sampled); allozyme markers are not sensitive enough to discriminate between F1, F2, Fn and backcross genotypes (except when at least four available polymorphic loci are applied to the Bayesian statistical method of Anderson and Thompson (2002, and per. com. E. Anderson)).

Nuclear RFLP’s - recently Billiones et al. (2004) developed a diagnostic nuclear restriction fragment length polymorphism (RFLP) analysis of the ITS gene region. With this method, some D. galeata specimens are incorrectly classified as D. cucullata or D. cucullata hybrids due to a mutation at the restriction site of MWO I. This problem could be solved with a new developed double restriction protocol (Skage et al. submitted). The disadvantage, however, of this technique is that only one species specific marker is available. If later generation of hybridization with backcrossing takes place in a population, hybrids cannot be distinguished with this technique.

RAPD’s - random amplified polymorphic DNA (RAPD) markers were developed (Ender and Schierwater 1993; Schierwater et al. 1994) to get an improved subdivision of hybrid classes. The RAPD technology enables investigation of numerous anonymous nuclear loci. However, results of a RAPD analysis depend strongly on PCR conditions, DNA quality, brand of Taq polymerase etc. Therefore it is difficult to repeat analyses between labs and samples.

Microsatellites - among the most popular genetic markers used nowadays (reviewed in Zane et al. 2002). Several microsatellite markers have been developed for this species complex (Ender et al. 1996; Brede et al. 2006). However, more testing has to be done with specimen from multiple lakes over a broad geographic area to evaluate which loci and alleles can be recommended as species specific.

Mitochondrial RFLP’s - a diagnostic mitochondrial RFLP analysis of the 16S rDNA (Schwenk et al. 1998). Disadvantage is that only maternal lineages can be discriminated.

It has been shown that the Daphnia community at times of sexual reproduction is not always representative for the entire growing season; in a temporary D. pulex pond population, for example, the population structure was significantly altered due to clonal selection during one season (Pfrender and Lynch 2000). Selection might be reinforced during parthenogenetic phases (i.e. clonal selection), because specific selective factors may act on entire genotypes rather than on individual levels (Vorburger et al. 2003). Clonal selection has been shown to be a very powerful evolutionary force, which

- 16 - Chapter 1 Introduction might lead to dramatic and fast population changes (Pfrender and Lynch 2000; Loxdale and Lushai 2003). In contrast, selective forces in Daphnia exist (fish and phantom midge larvae predation), which clearly act on the individual level rather than on the clonal level. Besides selection, assortative mating (e.g., Innes 1997) as well as hybridization can influence the outcome of sexual reproduction in Daphnia. Once ephippial eggs of Daphnia are produced they are subjected to various processes (Figure 2). Freshly produced ephippia can either become stuck to the surface tension or sink to the lake bottom (Stross 1966; Cáceres 1998). Buoyancy differences might be based on species-specific or clonal- specific external surface morphology of ephippia (Hebert 1995) and might influence the sedimentation process. Along a transect egg banks show, in general, a patchy distribution and a gradient with highest densities in the deepest part of the lake (see ‘sediment accumulation’ arrow in Figure 2) (reviewed by Brendonck and De Meester 2003). Further, the taxonomic affiliation of past populations can be obtained by taxonomic assignment of ephippial cases and hatched individuals as well as by direct DNA analysis. All these stages undergo degenerative processes, which can be demonstrated by low numbers of intact ephippia found in deep sediment layers (e.g., Limburg and Weider 2002), as well as by decreasing ability of older eggs to hatch (e.g., Weider et al. 1997) and DNA degeneration (e.g., Limburg and Weider 2002). Other circumstances impeding correct taxon identification are the limited number of diagnostic molecular markers available (Box 4) to analyze hybridizing Daphnia.

Thesis outline When I started the research described in this thesis, my goal was to disentangle factors that might explain the frequently observed hybrid dominance in hybridizing populations of the D. galeata-hyalina-cucullata species complex. I wanted to study microevolutionary and taxonomic changes over time caused by anthropogenic pollution by analysing preserved ephippial eggs from the sediment of a lake which underwent the process of eutrophication and oligotrophication. After reconstructing the historical ephippial distribution (Chapter 2), I analyzed hatchlings of archived and recently produced ephippial eggs, and quickly discovered that ephippial eggs do not match the pelagic Daphnia population perfectly (Chapter 3). From that moment on, I have focussed my research on processes causing this discrepancy, as well as on

- 17 - Introduction Chapter 1 factors that determine the Daphnia taxonomic composition in lakes: I investigated possible reproductive isolation mechanisms between hybridizing taxa (Chapter 4). As a final step, I was interested to know if taxon composition and hybrid success are habitat dependent. Therefore I analyzed the effect of environmental parameters with the main focus on the trophic change (i.e., anthropogenic pollution) among 43 lakes in Switzerland and Northern Italy (Chapter 5). All chapters are presented as manuscripts or already published papers and can be read on their own. This may result in overlaps between chapters when referring to literature data or results obtained in other chapters. This thesis concludes with Chapter 6, a synopsis of the previous chapters, a discussion of results in a broader perspective and suggestions for further research.

References Abernathy, K. 1994. The establishment of a hybrid zone between red and sika-deer (genus Cervus). Mol. Ecol. 3: 551-562. Allendorf, F. W., R. F. Leary, P. Spruell, and J. K. Wenburg. 2001. The problems with hybrids: setting conservation guidelines. Trends Ecol. Evol. 16: 613-622. Anderson, E. C., and E. A. Thompson. 2002. A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160: 1217-1229. Arnold, M. L. 1992. Natural hybridization as an evolutionary process. Annu. Rev. Ecol. Syst. 23: 237-261. Arnold, M. L. 1997. Natural hybridization and evolution, 1st ed. Oxford University Press. Arnold, M. L., E. K. Kentner, J. A. Johnston, S. Cornman, and A. C. Bouck. 2001. Natural hybridisation and fitness. Taxon 50: 93-104. Ayres, D. R., K. Zaremba, and D. R. Strong. 2004. Extinction of a common native species by hybridization with an invasive congener. Weed Technol 18: 1288-1291. Babik, W., J. M. Szymura, and J. Rafinski. 2003. Nuclear markers, mitochondrial DNA and male secondary sexual traits variation in a newt hybrid zone (Triturus vulgaris x T- montandoni). Mol. Ecol. 12: 1913-1930. Barton, N. H. 2001. The role of hybridization in evolution. Mol. Ecol. 10: 551-568. Barton, N. H., and G. M. Hewitt. 1985. Analysis of hybrid zones. Annu. Rev. Ecol. Syst. 16: 113-148. Barton, N. H., and G. M. Hewitt. 1989. Adaptation, speciation and hybrid zones. Nature 341: 497-503.

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Billiones, R., M. Brehm, J. Klee, and K. Schwenk. 2004. Genetic identification of Hyalodaphnia species and interspecific hybrids. Hydrobiologia 526: 43-53. Bilton, D. T., J. R. Freeland, and B. Okamura. 2001. Dispersal in freshwater invertebrates. Annu. Rev. Ecol. Syst. 32: 159-181. Bleeker, W., and H. Hurka. 2001. Introgressive hybridization in Rorippa (Brassicaceae): gene flow and its consequences in natural and anthropogenic habitats. Mol. Ecol. 10: 2013-2022. Boileau, M. G., and P. D. N. Hebert. 1991. Genetic consequences of passive dispersal in pond-dwelling copepods. Evolution 45: 721-733. Brede, N., A. Thielsch, C. Sandrock, P. Spaak, B. Keller, B. Streit, and K. Schwenk. 2006. Microsatellite markers for European Daphnia. Mol. Ecol. Notes 6: 536-539. Brendonck, L., and L. De Meester. 2003. Egg banks in freshwater zooplankton: evolutionary and ecological archives in the sediment. Hydrobiologia 491: 65-84. Britch, S. C., M. L. Cain, and D. J. Howard. 2001. Spatio-temporal dynamics of the Allonemobius fasciatus - A. socius mosaic hybrid zone: a 14-year perspective. Mol. Ecol. 10: 627-638. Bullini, L. 1985. Speciation by hybridization in animals. Boll. Zool. 52: 121-137. Burke, J. M., and M. L. Arnold. 2001. Genetics and the fitness of hybrids. Annu. Rev. Gen. 35: 31-52. Burke, J. M., M. R. Bulger, R. A. Wesselingh, and M. L. Arnold. 2000. Frequency and spatial patterning of clonal reproduction in Louisiana iris hybrid populations. Evolution 54: 137-144. Burke, J. M., S. E. Carney, and M. L. Arnold. 1998. Hybrid fitness in the Louisiana irises: Analysis of parental and F-1 performance. Evolution 52: 37-43. Cáceres, C. E. 1998. Interspecific variation in the abundance, production, and emergence of Daphnia diapausing eggs. Ecology 79: 1699-1710. Cáceres, C. E., and N. G. Hairston. 1998. Benthic-pelagic coupling in planktonic crustaceans: The role of the benthos. Arch. Hydrobiol. Spec. Issues Advanc. Limnol. 52: 163-174. Campbell, D. R., and N. M. Waser. 2001. Genotype-by-environment interaction and the fitness of plant hybrids in the wild. Evolution 55: 669-676. Carpenter, S. R., J. F. Kitchell, J. R. Hodgson, P. A. Cochran, J. J. Elser, M. M. Elser, D. M. Lodge, D. Kretchmer, X. He, and C. N. Vonende. 1987. Regulation of Lake Primary Productivity by Food Web Structure. Ecology 68: 1863-1876. Cousyn, C., L. De Meester, J. K. Colbourne, L. Brendonck, D. Verschuren, and F. Volckaert. 2001. Rapid local adaptation of zooplankton behavior to changes in predation pressure in absence of neutral genetic changes. Proc. Natl. Acad. Sci. U. S. A. 98: 6256-6260.

- 19 - Introduction Chapter 1

Cozzolino, S., A. M. Nardella, S. Impagliazzo, A. Widmer, and C. Lexer. 2006. Hybridization and conservation of Mediterranean orchids: Should we protect the orchid hybrids or the orchid hybrid zones? Biological Conservation 129: 14-23. De Meester, L., A. Gomez, B. Okamura, and K. Schwenk. 2002. The Monopolization Hypothesis and the dispersal-gene flow paradox in aquatic organisms. Acta Oecologica 23: 121-135. De Stasio, B. T., Jr. 1989. The seed bank of a freshwater crustacean: Copepodology for the plant ecologist. Ecology 70: 1377-1389. Dobzhansky, T. 1940. Speciation as a stage in evolutionary divergence. Am. Nat. 74: 312- 321. Dowling, T. E., and C. L. Secor. 1997. The role of hybridization and introgression in the diversification of animals. Annu. Rev. Ecol. Syst. 28: 593-619. Edmands, S. 2002. Does parental divergence predict reproductive compatibility? Trends Ecol. Evol. 17: 520-527. Edmondson, W. T., and A. H. Litt. 1982. Daphnia in Lake Washington. Limnol. Oceanogr. 27: 272-293. Ellstrand, N. C., and K. A. Schierenbeck. 2000. Hybridization as a stimulus for the evolution of invasiveness in plants? Proc. Natl. Acad. Sci. U. S. A. 97: 7043-7050. Emms, S. K., and M. L. Arnold. 1997. The effect of habitat on parental and hybrid fitness: transplant experiments with Louisiana irises. Evolution 51: 1112-1119. Ender, A., and B. Schierwater. 1993. Identification of nuclear markers in Daphnia hybrid species complexes using random amplified polymorphic DNA (RAPD). Verhandlungen der Deutschen Zoologischen Gesellschaft 86.1: 45. Ender, A., K. Schwenk, T. Stadler, B. Streit, and B. Schierwater. 1996. RAPD identification of microsatellites in Daphnia. Mol. Ecol. 5: 437-441. Epifanio, J., and D. Philipp. 2000. Simulating the extinction of parental lineages from introgressive hybridization: the effects of fitness, initial proportions of parental taxa, and mate choice. Reviews in Fish Biology and Fisheries 10: 339-354. Eriksen, B., and M. H. Topel. 2006. Molecular phylogeography and hybridization in members of the circumpolar Potentilla sect. Niveae (Rosaceae). Am. J. Bot. 93: 460- 469. Ferdy, J. B., and F. Austerlitz. 2002. Extinction and introgression in a community of partially cross-fertile plant species. Am. Nat. 160: 74-86. Frey, D. 1982. Contrasting strategies of gamogenesis in northern and southern populations of Cladocera. Ecology 63: 223-241. Gießler, S. 1997. Analysis of reticulate relationships within the Daphnia longispina species complex. Allozyme phenotype and morphology. J. Evol. Biol. 10: 87-105.

- 20 - Chapter 1 Introduction

Hairston, N. G., W. Lampert, C. E. Cáceres, C. L. Holtmeier, L. J. Weider, U. Gaedke, J. M. Fischer, J. A. Fox, and D. M. Post. 1999a. Lake ecosystems - Rapid evolution revealed by dormant eggs. Nature 401: 446. Hairston, N. G., L. J. Perry, A. J. Bohonak, M. Q. Fellows, C. M. Kearns, and D. R. Engstrom. 1999b. Population biology of a failed invasion: Paleolimnology of Daphnia exilis in upstate New York. Limnol. Oceanogr. 44: 477-486. Hall, R. J., A. Hastings, and D. R. Ayres. 2006. Explaining the explosion: modelling hybrid invasions. Proc. R. Soc. Lond. B 273: 1385-1389. Harrison, R. G. 1985. Pattern and process in a narrow hybrid zone. Heredity 56: 337-349. Hebert, P. D. N. 1985. Interspecific hybridization between cyclic parthenogens. Evolution 39: 216-220. Hebert, P. D. N. 1988. Genetics of Daphnia, p. 439-460. In R. H. Peters and R. De Bernardi [eds.], Daphnia. Memorie dell'instituto di idrobiologia. Hebert, P. D. N. 1995. The Daphnia of Northern America: An illustrated fauna. CD-ROM distributed by the author. Hewitt, G. M. 2001. Speciation, hybrid zones and phylogeography - or seeing genes in space and time. Mol. Ecol. 10: 537-549. Howard, D. J. 1986. A zone of overlap and hybridization between two ground cricket species. Evolution 40: 34-43. Howard, D. J. 1993. Reinforcement: Origin, dynamics, and fate of an evolutionary hypothesis, p. 46-69. In R. G. Harrison [ed.], Hybrid zones and the evolutionary process. Oxford University Press. Howard, D. J., G. L. Waring, C. A. Tibbets, and P. G. Gregory. 1993. Survival of hybrids in a mosaic hybrid zone. Evolution 47: 789-800. Innes, D. J. 1997. Sexual reproduction of Daphnia pulex in a temporary habitat. Oecologia 111: 53-60. Innes, D. J., and D. R. Singleton. 2000. Variation in allocation to sexual and asexual reproduction among clones of cyclically parthenogenetic Daphnia pulex (Crustacea : Cladocera). Bio. J. Linn. Soc. 71: 771-787. Jankowski, T., and D. Straile. 2003. A comparison of egg-bank and long-term plankton dynamics of two Daphnia species, D. hyalina and D. galeata: Potentials and limits of reconstruction. Limnol. Oceanogr. 48: 1948-1955. Kerfoot, W. C., and L. J. Weider. 2004. Experimental paleoecology (resurrection ecology): Chasing Van Valen's Red Queen hypothesis. Limnol. Oceanogr. 49: 1300-1316. Key, K. H. L. 1968. Concept of stasipatric speciation. Systematic Zoology 17: 14-&. Korpelainen, H. 1989. Sex ratio of the cyclic parthenogen Daphnia magna in a variable environment. Z. zool. Syst. Evolut. forsch. 27: 310-316. Levin, D. A. 2004. The ecological transition in speciation. New Phytol. 161: 91-96.

- 21 - Introduction Chapter 1

Levin, D. A., J. Franciscoortega, and R. K. Jansen. 1996. Hybridization and the extinction of rare plant species. Conserv. Biol. 10: 10-16. Lexer, C., M. E. Welch, O. Raymond, and L. H. Rieseberg. 2003. The origin of ecological divergence in Helianthus paradoxus (Asteraceae): Selection on transgressive characters in a novel hybrid habitat. Evolution 57: 1989-2000. Limburg, P. A., and L. J. Weider. 2002. “Ancient” DNA in a microcrustacean resting egg bank can serve as a paleolimnological database. Proc. R. Soc. Lond. B 269: 281-287. Loxdale, H. D., and G. Lushai. 2003. Rapid changes in clonal lines: the death of a 'sacred cow'. Bio. J. Linn. Soc. 79: 3-16. Mallet, J. 2005. Hybridization as an invasion of the genome. Trends Ecol. Evol. 20: 229- 237. Marshall, J. L., M. L. Arnold, and D. J. Howard. 2002. Reinforcement: the road not taken. Trends Ecol. Evol. 17: 558-563. Mellors, W. K. 1975. Selective predation of ephippial Daphnia and the resistance of ephippial eggs to digestion. Ecology 56: 974-980. Moore, W. S. 1977. An evaluation of narrow hybrid zones in vertebrates. Q. Rev. Biol. 52: 263-277. Moore, W. S., and W. D. Koenig. 1986. Comparative reproductive success of yellow- shafted, red-shafted and hybrid flickers across a hybrid zone. Auk 103: 42-51. Nosil, P., T. H. Vines, and D. J. Funk. 2005. Perspective: Reproductive isolation caused by natural selection against immigrants from divergent habitats. Evolution 59: 705-719. Pfrender, M. E., and M. Lynch. 2000. Quantitative genetic variation in Daphnia: Temporal changes in genetic architecture. Evolution 54: 1502-1509. Rhymer, J. M., and D. Simberloff. 1996. Extinction by hybridization and introgression. Annu. Rev. Ecol. Syst. 27: 83-109. Rieseberg, L. H. 1997. Hybrid origins of plant species. Annu. Rev. Ecol. Syst. 28: 359-389. Rieseberg, L. H., and S. E. Carney. 1998. Plant hybridization. New Phytol. 140: 599-624. Schierwater, B., A. Ender, K. Schwenk, P. Spaak, and B. Streit. 1994. The evolutionary ecology of Daphnia, p. 495-508. In B. Schierwater, B. Streit, G. Wagner and R. DeSalle [eds.], Molecular Ecology and Evolution: Approaches and Applications. Birkhäuser Verlag. Schwartz, S. S., and P. D. N. Hebert. 1987. Methods for activation of the resting eggs of Daphnia. Freshwat. Biol. 17: 373-379. Schwenk, K., A. Sand, M. Boersma, M. Brehm, E. Mader, D. Offerhaus, and P. Spaak. 1998. Genetic markers, genealogies and biogeographic patterns in the cladocera. Aquatic Ecology 32: 37-51. Schwenk, K., and P. Spaak. 1995. Evolutionary and ecological consequences of interspecific hybridization in cladocerans. Experientia 51: 465-481.

- 22 - Chapter 1 Introduction

Schwenk, K., and P. Spaak. 1997. Ecological and genetics of interspecific hybridization in Daphnia, p. 199-229. In B. Streit, T. Städler and C. M. Lively [eds.], Evolutionary ecology of freshwater animals. Birkhäuser Verlag. Seehausen, O. 2004. Hybridization and adaptive radiation. Trends Ecol. Evol. 19: 198-207. Skage, M., A. Hobæk, Š. Ruthová, B. Keller, A. Petrusek, J. Seća, and P. Spaak. submitted. Intragenomic sequence variation necessitates a new genetic method to distinguish species and hybrids in the Daphnia longispina complex. submitted to Hydrobiologia. Spaak, P. 1996. Temporal changes in the genetic structure of the Daphnia species complex in Tjeukemeer, with evidence for backcrossing. Heredity 76: 539-548. Spaak, P., and J. R. Hoekstra. 1995. Life history variation and the coexistence of a Daphnia hybrid with its parental species. Ecology 76: 553-564. Stebbins, G. L. 1950. Variation and evolution in plants. Columbia University Press. Stross, R. G. 1966. Light and temperature requirements for diapause development and release in Daphnia. Ecology 47: 368-374. Swenson, N. G. 2005. Gis-based niche models reveal unifying climatic mechanisms that maintain the location of avian hybrid zones in a North American suture zone. J. Evol. Biol. 19: 717-725. Taylor, D. J., and P. D. N. Hebert. 1992. Daphnia galeata mendotae as a cryptic species complex with interspecific hybrids. Limnol. Oceanogr. 37: 658-665. Turelli, M., N. H. Barton, and J. A. Coyne. 2001. Theory and speciation. Trends Ecol. Evol. 16: 330-343. Vorburger, C., P. Sunnucks, and S. A. Ward. 2003. Explaining the coexistence of asexuals with their sexual progenitors: no evidence for general-purpose genotypes in obligate parthenogens of the peach-potato aphid, Myzus persicae. Ecol. Lett. 6: 1091-1098. Weider, L. J. 1993. Niche breadth and life history variation in a hybrid Daphnia complex. Ecology 74: 935-943. Weider, L. J., and A. Hobaek. 2003. Glacial refugia, haplotype distributions, and clonal richness of the Daphnia pulex complex in arctic Canada. Mol. Ecol. 12: 463-473. Weider, L. J., W. Lampert, M. Wessels, J. K. Colbourne, and P. Limburg. 1997. Long-term genetic shifts in a microcrustacean egg bank associated with anthropogenic changes in the Lake Constance ecosystem. Proc. R. Soc. Lond. B 264: 1613-1618. Wirtz, P. 1999. Mother species-father species: unidirectional hybridization in animals with female choice. Animal Behaviour 58: 1-12. Wolf, D. E., N. Takebayashi, and L. H. Rieseberg. 2001. Predicting the risk of extinction through hybridization. Conserv. Biol. 15: 1039-1053. Wolf, H. G. 1987. Interspecific hybridization between Daphnia hyalina, D. galeata and D. cucullata and seasonal abundance of these species and their hybrids. Hydrobiologia 145: 213-217.

- 23 - Introduction Chapter 1

Wolf, H. G., and M. A. Mort. 1986. Interspecific hybridization underlies phenotypic variability in Daphnia populations. Oecologia 68: 507-511. Wolinska, J., B. Keller, K. Bittner, S. Lass, and P. Spaak. 2004. Do parasites lower Daphnia hybrid fitness? Limnol. Oceanogr. 49: 1401-1407. Zane, L., L. Bargelloni, and T. Patarnello. 2002. Strategies for microsatellite isolation: a review. Mol. Ecol. 11: 1-16.

- 24 -

CHAPTER 2

Ephippia and Daphnia abundances under changing trophic conditions with H. R. Bürgi, M. Sturm, and P. Spaak Verhandlungen Internationale Vereinigung für Theoretische und Angewandte Limnologie 28, 851-855 (2002)

Introduction Many lakes that were overloaded with nutrients and became hypertrophic in the 1960s and 1970s have since gone through a process of oligotrophication. In spite of this, they are still classified as eutrophic (Ruggiu et al. 1998; Van Der Molen et al. 1998). Eutrophic conditions have fundamentally changed the biotic and abiotic conditions in these lakes. For example, deeper layers became anoxic in summer, leaving a reduced stratum for plankton and fish. Since nutrients were available in larger quantities, plankton increased in density. High nutrient density can result in algal blooms but also in the collapse of algae-feeding zooplankton populations, due to lack of food. Daphnia play an important role in lake food webs (Manca and Ruggiu 1998). They link the primary production of planktonic algae with vertebrate predators. Daphnids can attain high biomass in a short time period because of parthenogenetic reproduction. For this reason, they are an important food source for planktivorous fish in many temperate lakes. When food availability is poor, however, as happens regularly in eutrophic lakes during summer, daphnids start to reproduce sexually. Sexual reproduction results in the production of dormant stages, termed ephippia. Ephippia sink, (influenced by gravity, wind and current) to the lake bottom, where they accumulate in the sediment, forming a so-called ‘egg-bank’ (Hairston 1996). Daphnids are short- lived organisms but dormant ephippia can survive for decades (Carvalho and Wolf 1989). If suitable conditions occur again, daphnids can hatch from the ephippia.

- 25 - Egg bank Chapter 2

In eutrophic lakes, where the sediment is varved due to seasonal changes of processes of particle production (Sturm and Lotter 1995), the Daphnia egg- bank can be dated relatively easily (Ambühl 1994). As such, it is possible to compare the number of ephippia in a sediment layer with biotic and abiotic conditions in the lake at that time (if those conditions were monitored). Egg- bank dynamics were investigated at three stations in Lake Greifensee, Switzer- land, with the aim of quantifying the temporal and spatial variability in the distribution of Daphnia ephippia in the lake. Furthermore, the hypothesis that greater ephippial densities occurred in sediment strata during the hypertrophic period of the lake was tested. Because of the strong fluctuations in living conditions during this phase, daphnids were expected to produce a greater number of resting eggs. For this study, long-term plankton records collected by one of the present authors (Bürgi, H. R. unpubl.) were compared with results from sediment cores.

Figure 1. Schematic map (modified from Wan et al. 1987) of Lake Greifensee with the three sampling stations, the lake outlet (Glatt) and the two main lake inlets (Aa-Bach, Mönchaltdorfer-Aa).

Material and methods Greifensee (Switzerland) is a highly eutrophic midland lake with a surface area of 8.5 km2 and a volume of 150 × 106 m3. Its average depth is 17.7 m, with a maximum depth of 33 m. Total phosphorus values in the lake in February 2000 were 79.3 ȝg L-1 and have declined sharply during the last 20 years. Two main rivers (Aa-Bach, Möchaltdorfer-Aa) flow into the lake, while one lake outlet (Glatt) drains the system (Fig. 1). Only the larger Aa-Bach seems to create a noticeable flow-through towards the lake outlet. The first major step of eutrophication of the lake started in the beginning of the last century (Guyer 1910; Mühlethaler et al. 1993). After the next peak of eutrophication in 1973

- 26 - Chapter 2 Egg bank

-1 (Ptot February = 535 ȝg L ), restoration measures were started. Since then, Ptot values have decreased but the lake is still eutrophic (Mühlethaler et al. 1993).

80 total Phosphorus 400 60 all Daphnids (A) 300 40 200

20 100

0 0 0.6 400 0.5 total Phosphorus ephippia (μg/L) P total 300 0.4 males (B) total Daphnids litre per total Daphnids 0.3 200 0.2 100 0.1 0.0 0 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 year Figure 2. Abundance of Daphnia and total phosphorus (1975 – 2000). (A) All parthenogenetic females (juveniles, adults with eggs and adults without out eggs). (B) Sexual Daphnia (males, ephippial females and floating ephippia).

Quantitative zooplankton samples have been collected every month since 1975 with a 95 ȝm double closing net (Bürgi 1983) and preserved in 4% formalin. Samples were taken from the deepest point of the Greifensee (Station 2, Fig. 1) (Bürgi et al. 1985). Adult daphnids without eggs, adult daphnids with eggs, juvenile daphnids, and ephippial females, together with floating ephippia, were enumerated separately (Bürgi et al. 1999). Two Daphnia species co-occur in the Greifensee (D. galeata and D. hyalina). These species hybridize and the hybrids probably produce back-crosses (Spaak et al. 2000). Because of many intermediate morphological forms, daphnids were not identified to species level. Several studies have dated the varved Greifensee sediments and calculated sedimentation rates of at approximately 0.14 g cm-2year-1 (Wan et al. 1987; Ambühl 1994). The coring technique of Ambühl (1985) was used to take two types of cores at three locations in the lake. In 1999, two cores were taken at station 2; in 2000, three additional cores at stations 1, 2 and 3 were collected (Fig. 1). In 2000, two cores (11.2-cm inner core diameter) were taken per station. One core was sliced vertically in two parts, using a sheet of glass, to take pictures for later identification of different varves. The other was cut in 1.4-cm thick slices using Petri-dishes (8.9-cm inner diameter), which excluded

- 27 - Egg bank Chapter 2 the outer edge of sediment; which was smudged along the sampling pipe. Petri-dishes were pressed into the sediment and cut beneath each dish with a metal plate, numbered, wrapped with aluminium foil and stored until use in a refrigerator (dark, 4 °C). One-quarter of each sample from cores 1 and 3 was cut out of the Petri-dishes under dimmed light for dating. Theses sediment samples were dried at 60 °C, ground with a mortar and used for dating by isotope measurements with a CANBERRA well-type detector. Additionally, the cores were dated using the varve chronology of Wan et al. (1987) and recent dating information from station 2, where a new varve chronology was available from a freeze core taken in 1994 (Sturm, M. unpubl., Lotter et al. 1997). The remainder of each sampling slice was sieved through a 150-ȝm mesh sieve to remove as much fine sediment as possible. Ephippia were removed with forceps from the remaining sample using a stereo microscope and counted.

Results and discussion The long-term study of the zooplankton community in Greifensee shows that the Daphnia population underwent apparent abundance changes during the last 25 years (Fig. 2A). Parallel to the decrease in total phosphorus concentrations, striking Daphnia density changes observed in the beginning of the period were reduced, except one large peak in 1997. The marked fluctuations in population density are typical for an eutrophic lake, based on algae growth alternating with overgrazing by herbivore zooplankton such as daphnids (Bürgi 1994; Carpenter and Cottingham 1997). The abundance peak in 1997 could be explained by the fact that the lake is still eutrophic and far from its natural condition. Densities of ephippial females (Fig. 2B) do not show the same pattern as parthenogenetic Daphnia over the last 25 years. Daphnia males were recorded since 1995 (Fig. 2B), a peak (0.09 L-1) was recorded in 1997 when high densities of parthenogenetic females were also present. However, no peak in ephippial females occurred. It is known that the production of males and ephippial females is partly induced by different stimuli (Kleiven et al. 1992). Furthermore, the chance of an ephippial female mating with a male will increase if the density of males is relatively high. The present results show that the number of ephippia produced did not correlate with Daphnia density (R2 = 0.19; correlation not shown).

- 28 - Chapter 2 Egg bank

) Sediment 2 16 ·m 3 12

4 2

ephippia (N·10 R = 0.91

0 100 Lake Figure 3. Sediment: ephippia abundance in ) 80 3 -2 3 R2 = 0.02 the sediment (10 m ; mean of two cores 60 taken at station 2) correlated with its 40 corresponding total phosphorus value (ȝg L-1; R2 = 0.9; average per year). Lake: ephippia (N·m 20 yearly mean numbers of ephippial females 0 and floating ephippia correlated with the 0 100 200 300 400 -1 yearly mean Ptot values (ȝg L ) total P (μg L)

The total number of pelagic ephippial Daphnia found per year did not significantly correlate with the concentration of Ptot during spring in the Greifensee for the last 25 years (R2 = 0.02; Fig. 3). This was unexpected, but could be a result of insufficient sampling. The analysis was completed on monthly zooplankton samples taken at one station (2) over the 25 years. Many studies have showed that zooplankton are not distributed equally in a lake; distributions are rather patchy (Bürgi et al. 1993). Ephippia may also have drifted to the shore of the lake with the wind. Therefore, the number of ephippia in the water column at station 2 could be an underestimate of the total number of ephippia produced. A better estimate for the total number of ephippia produced per year is the ephippial density in the sediment, particularly in the deeper anoxic part of the lake where the sediment is not disturbed by benthic fauna. The core taken at the deepest part of the lake (core 2) is varved until the most recent layers (Fig. 4). The varved period in core 1 and the varved period in core 3 have almost the same thickness, but they do not seem to originate from the same period (Fig. 4). Core 1 shows varves in the deeper part of the core, while core 3 displays a homogeneous layer without visible laminations at this depth of the core. These differences may be explained by the different locations of the cores, which show distinctly different sedimentation rates. Thus, sediment rates, determine by 137Cs, were comparable at station 2 and 3 (3.3 mm year-1), but were much higher at station 1 (5.8 mm year-1) (Fig. 4).

- 29 - Egg bank Chapter 2

Figure 4. Varved sediment cores dated by137Cs (Bq kg-1) and by varve counting from the three different stations in Greifensee (137Cs peaks mark the bomb fallout of 1963 and the Chernobyl fallout in 1986, respectively). Core 1 was taken at station 1, close to the lake inlet of the Mönchaltdorfer-Aa. Core 2 was taken at station 2 at the deepest point of the lake. Core 3 was taken at station 3 close to the outlet of the Lake (River Glatt).

A better estimate for the total number of ephippia produced per year is the ephippia density in the sediment, particularly in the deeper anoxic part of the lake, the sediment is not disturbed by benthic fauna. The core taken at the deepest part of the lake (core 2) is varved until the most recent layers (Fig. 4). The varved period in core 1 and the varved period in core 3 have almost the same thickness, but they do not seem to originate from the same period (Fig. 4). Core 1 shows varves in the deeper part of the core, while core 3 displays a homogeneous layer without visible laminations at this depth of the core. These differences may be explained by the different locations of the cores, which show distinctly different sedimentation rates. Thus, sediment rates, determine by 137Cs, were comparable at station 2 and 3 (3.3 mm year-1), but were much higher at station 1 (5.8 mm year-1) (Fig. 4). These different sedimentation rates may influence the different ephippial distributions within the sediment cores of the three stations (Fig. 5). In the core taken close to the lake outlet (station 3) almost no ephippia were found below 18 cm, whereas Daphnia ephippia were found up to 26 cm (the deepest layer

- 30 - Chapter 2 Egg bank analysed) at the other two stations. The absence of Daphnia ephippia in the deepest layers of core 3 may be caused by erosion. Nonetheless, a general distribution pattern can be seen in all three cores. The youngest and oldest sediment layers contained considerably fewer ephippia than the intermediate layers. When the mean ephippial distribution of the two cores taken at station 2 is correlated with the annual mean of total phosphorus concentration during the last 25 years, a high linear correlation emerged (R2 = 0.91; Fig. 3A). This result suggested that the history of sexual reproduction in Daphnia can be assessed better with ephippial densities from the sediment than with monthly or bi-weekly plankton samples taken at one location in the lake. The study also showed that daphnids produce the most ephippia during eutrophic conditions in a lake.

2 M-Aa (1) 6 10 14 18 22 26

2 2 deepest point (2) 10 6 19 10 30 42 14 57 18 78 99 22 123 sediment depth (cm)sediment depth 26 147 sediment age (years) Figure 5. Ephippial distribution in the 2 Glatt (3) 6 sediment at the three sampling stations; 10 Mönchaltdorfer-Aa (M-Aa, station 1); 14 deepest point in the lake (station 2); Glatt 18 (station 3). Sediment age on the right 22 y-axis for station 2 was determined with 26 the varve counting method, calibrated from 0 30 60 90 120 150 # ephippia (m2*103) (Wan et al. 1987) .

Conclusions In this study, it has been shown that the trophic stage of Greifensee strongly affected the production of Daphnia ephippia, based on their abundance in dated sediment cores. The higher the total phosphorus concentration in the lake, the greater the accumulation of ephippia. Local conditions in the lake, like wind and current, exert an important influence on the ephippial distribution in the sediment, and thus their hatching success. It is known that different Daphnia species and genotypes hatch under different environmental

- 31 - Egg bank Chapter 2 conditions (Schwartz and Hebert 1987). Moreover, the entire Daphnia population can react to these environmental changes. Further research is needed to determine if different Daphnia clones or species hatch at the different sampling stations in Greifensee.

Acknowledgements The authors thank Heinrich Bührer who has put the long-term data of total phosphorus at our disposal. Furthermore, we thank Chris Robinson for useful suggestions on an earlier version of the manuscript. This work was supported by a grant from the Swiss Federal Office for Education and Science (no. 97.0040) to PS, and was part of the European Commission’s Environment and Climate Programme which is part of the project network WatER (Wetland and Aquatic Ecosystem Research).

References Ambühl, H. 1985. Technik der Präperation und Darstellung von Sedimentkernen mit grossem Querschnitt. Schweiz. Z. Hydrol. 47: 249-256. Ambühl, H. 1994. Die Feinstruktur jüngster Sedimente von Seen verschiedenen Trophiegrades und von Seen in technischer Sanierung. Limnol. Ber. Donau 2: 101- 126. Bürgi, H. R. 1983. Eine neue Netzgarnitur mit Kipp-Schliessmechanismus für quantitative Zooplanktonfänge in Seen. Schweiz. Z. Hydrol. 45: 505-507. Bürgi, H. R. 1994. Seenplankton und Seensanierung in der Schweiz. Limnol. Ber. Donau 2: 71-100. Bürgi, H. R., J. J. Elser, R. C. Richards, and C. R. Goldman. 1993. Zooplankton patchiness in Lake Tahoe and Castle Lake. Verh. Internat. Verein. Limnol. 25: 378-382. Bürgi, H. R., C. Heller, S. Gaebel, N. Mookerji, and J. V. Ward. 1999. Strength of coupling between phyto- and zooplankton in Lake Lucerne (Switzerland) during phosphorus abatement subsequent to a weak eutrophication. J. Plankton Res. 21: 485-507. Bürgi, H. R., P. Weber, and H. Bachman. 1985. Seasonal variations in the trophic structure of phyto- and zooplankton communities in lakes in different trophic states. Schweiz. Z. Hydrol. 47: 197-224. Carpenter, S. R., and K. L. Cottingham. 1997. Resilience and restoration of lakes. Biological Abstracts Vol. 104, Iss. 10, Ref. 143453. 1: Available from the Internet. URL: http://www.consecol.org/vol1/iss1/art2.

- 32 - Chapter 2 Egg bank

Carvalho, G. R., and H. G. Wolf. 1989. Resting eggs of lake-Daphnia. I. Distribution, abundance and hatching of eggs collected from various depths in lake sediments. Freshwat. Biol. 22: 459-470. Guyer, O. 1910. Beiträge zur Biologie des Greifensees mit besonderer Berücksichtigung der Saisonvariation von Ceratium hirundinella. Ph.D. ETH. Hairston, N. G. 1996. Zooplankton egg banks as biotic reservoirs in changing environments. Limnol. Oceanogr. 41: 1087-1092. Kleiven, O. T., P. Larsson, and A. Hobæk. 1992. Sexual reproduction in Daphnia magna requires three stimuli. Oikos 65: 197-206. Lotter, A. F., M. Sturm, J. L. Teranes, and B. Wehrli. 1997. Varve formation since 1885 and high-resolution varve analyses in hypertrophic Baldeggersee (Switzerland). Aquat. Sci. 59: 304-325. Manca, M., and D. Ruggiu. 1998. Consequences of pelagic food-web changes during a long-term lake oligotrophication process. Limnol. Oceanogr. 43: 1368-1373. Mühlethaler, E., G. Gelpke, S. Schneider, and K. König Urmi. 1993. Der Greifensee: Eine Dokumentation. Verband zum Schutze des Greifensees Uster. Ruggiu, D., G. Morabito, P. Panzani, and A. Pugnetti. 1998. Trends and relations among basic phytoplankton characteristics in the course of the long-term oligotrophication of Lake Maggiore (Italy). Hydrobiologia 370: 243-257. Schwartz, S. S., and P. D. N. Hebert. 1987. Methods for activation of the resting eggs of Daphnia. Freshwat. Biol. 17: 373-379. Spaak, P., L. Eggenschwiler, and H. Bürgi. 2000. Genetic variation and clonal differentiation in the Daphnia population of the Greifensee, a pre-alpine Swiss lake. Verh. Internat. Verein. Limnol. 27: 1919-1923. Sturm, M., and A. F. Lotter. 1995. Lake sediments as environmental archives - a means of distinguishing natural events from human activity. EAWAG news 38E: 6-9. Van Der Molen, D. T., R. Portiele, P. C. M. Boers, and L. Lijklema. 1998. Changes in sediment phosphorus as a result of euthrophication and oligotrophication in Lake Veluwe, The Netherlands. Water Res. 32: 3281-3288. Wan, G. J. and others 1987. Natural (210Pb, 7Be) and fallout (137Cs, 239,240Pu, 90Sr) radionuclides as geochemical tracers of sedimentation in Greifensee, Switzerland. Chem. Geol. 63: 181-196.

- 33 -

CHAPTER 3

Non-random sexual reproduction and diapausing egg production in a Daphnia hybrid species complex with P. Spaak Limnology and Oceanography 49, 1393–1400 (2004)

Abstract Sexual reproduction in Daphnia results in the production of diapausing eggs, which are enclosed in a structure called an ephippium. Ephippia, accumulated by sedimentation, can be preserved for decades and offer the opportunity for microevolutionary studies as well as the study of former pelagic populations. In a Swiss subalpine lake (Greifensee), we studied the genetic structure of the pelagic Daphnia galeata × hyalina hybrid species complex. We examined sexual females, males, and ephippial production. Eggs from ephippia were hatched, and the genotypes and taxa composition of all daphnids were determined using four polymorphic allozyme loci, two of which are each diagnostic for D. galeata and D. hyalina. We found significant differences between the genetic composition and the backcross level of pelagic asexual females, sexual females, males, and ephippial eggs (Daphnia hatchlings). The asexual daphnids were dominated by hybrids. In contrast, sexual females, especially Daphnia hatched from ephippial eggs, are dominated by D. galeata. We conclude that hybrid Daphnia have a lower sexual reproductive success than the parental D. galeata. The recent hybrid dominance suggests that D. galeata that hatch from diapausing eggs are not able to alter the pelagic population. The genotypic class composition of the diapausing eggs does not reflect the extant pelagic population; therefore, Daphnia diapausing egg banks do not always represent the past lake taxon structure.

- 35 - Sexual reproduction Chapter 3

Introduction Lake sediments are ideal archives to examine climatic changes (e.g., Lotter et al. 1998), ancient industrialization (Branvall et al. 2001), or changes in species patterns through time. Diverse organic remains (seeds, diatoms, pollen, and remains of Cladocera; Lotter et al. 1998) enable the investigation of processes in the past. Remains of Cladocera have proven useful for paleolimnological studies (Frey 1974), reconstructing lake plankton communities of 10,000 yr ago (Duigan and Birks 2000). Evolutionary changes in species composition and morphology or possible hybridization events can be documented (e.g. Hofmann 1991) using residuals of mucrons, postabdominal claws, and diapausing eggs. Recently, researchers have begun to examine diapausing eggs of zooplankton by hatching them (Hairston et al. 1999; Kerfoot et al. 1999) and to study their genetic makeup by directly genotyping the eggs (Duffy et al. 2000; Reid et al. 2000; Limburg and Weider 2002). Genetic analysis of sexually produced diapausing eggs of Daphnia, either directly or indirectly (by genotyping the hatchlings), enables the study of past genetic changes in plankton populations. Sexually produced diapausing eggs are encapsulated in a protective envelope called an ephippium. After a dormant phase, eggs hatch if they are exposed to proper hatching stimuli. Although the hatching process is not fully understood, light and elevated temperature may play a role in breaking dormancy (Schwartz and Hebert 1987). Ex-ephippial hatchlings from lake sediments have shown that the genetic composition of populations can change with time (e.g., Weider et al. 1997). Limburg and Weider (2002) found significant shifts between the genetic makeup of recent ephippia and those produced >150 yr ago in the Daphnia longispina group. In contrast, Cousyn et al. (2001) studied a relatively young Daphnia magna diapausing egg bank using microsatellites and found no genetic shift in time. The advantage of analyzing eggs directly is that postfertilization selection influences (e.g., selective hatching or mortality) can be avoided. Such an approach allows the study of genetic changes for much longer time frames. On the other hand, life-history experiments can be conducted on ex-ephippial hatchlings (Hairston et al. 2001). An important question remaining is how well diapausing egg banks represent former pelagic populations. A general mismatch between egg banks and extant populations of cyclical parthenogens has been found for rotifers (Gomez and Carvalho 2000) and daphnids (e.g., Weider et al. 1997; Cáceres 1998); however, Weider et al. (1997) used only one species-specific genetic marker. Such an approach neglects the complex system of hybridization and

- 36 - Chapter 3 Sexual reproduction backcrossing in hybrid Daphnia swarms. In many European midland lakes, parental species and hybrids (F1 hybrids and backcrosses) co-occur (Spaak

1996). In some populations, F1 hybrids may numerically dominate one or both parental species (e.g., Schwenk and Spaak 1995). The accurate classification of individuals is difficult in such complex systems with many generations of hybridization and backcrossing. Morphological, nuclear, and allozyme species- specific markers have been developed and combined to try to solve this problem (e.g., Gießler 1997). Campton and Utter (1985) described a method to classify parental and hybrid taxa using genetic data without the assumption of unique alleles. Anderson and Thompson (2002) used a combination of diagnostic and non-diagnostic genetic markers to compute the probability of misclassification of particular individuals. The cyclical parthenogenesis of Daphnia in large European midland lakes offers the possibility for year-long clonal propagation, thus reducing the effect of sexual reproduction. However, Spaak (1997) found that hybrids exhibit high levels of genetic variation, and Jankowski (2002) showed that hybrids produce fertile sexual individuals. Nonetheless, parental species remain distinct, even when hybrids and parentals occur together (Spaak 1996). Little is known about the fate of hybrid or backcrossed diapausing eggs compared with intraspecific ones, although Schwenk et al. (2001) found differences in hatching success between experimental intra- and interspecific crosses, as well as backcrosses between Daphnia galeata and Daphnia cucullata. We investigated the processes that determine the genetic composition of diapausing eggs in hybridizing Daphnia species. We examined whether the various hybrid classes engage equally in sexual reproduction and how well diapausing eggs and ex-ephippial hatchlings from deep sediment reflect former Daphnia populations in hybrid species complexes. We analyzed the genetic composition of pelagic sexual and asexual Daphnia in detail, as well as the genetic composition of ex-ephippial neonates hatched from recently produced ephippia. Finally, we compared these data with the genetic pattern of laboratory hatchlings from various sediment depths, to determine whether hybrid taxon dominance in recent populations is represented in egg banks.

- 37 - Sexual reproduction Chapter 3

100 80 60 40

proportion (%) proportion 20 A 0

) 500 -1 F2 Phyalina 400 BPgaleata BPhyalina

1000 Pgaleata F1 -2 300 200 100 B individuals (m individuals 0 100 80 60 backcrosses F1 40 Pgaleata

proportion (%) proportion 20 C 0 Jun 02 Jun 01 Jun 00 Jun 98 Jun 99 Sep 01 Sep Sep 00 Sep Sep 98 Sep Sep 99 Sep Dec 01 Dec Dec 00 Dec Dec 98 Dec Dec 99 Dec Mar 02 Mar 01 Mar 99 Mar 00 Mar 98

Figure 1. Genealogical classes in asexual daphnids of Greifensee. (A) Proportional distribution (class identification according to Nason and Ellstrand (1993): Pgaleata, Phyalina, F1, F2, backcrossed [BP] galeata, and BPhyalina), with arrows marking dates when sexual females could be sampled. (B) Effective abundance based on quantitative sampling counts and genealogical class proportions (see panel A). (C) Proportion of three genealogical classes according, to Anderson and Thompson (2002).

Material and methods Study site – The study was performed in a eutrophic Swiss subalpine lake (Greifensee). Greifensee has a surface area of 8.5 km2, a volume of 150 × 106 m3, and an average depth of 17.7 m, with a maximum depth of 33 m. Total phosphorus values in the lake in February 2000 were 79.3 μg L-1 and have declined sharply the last 25 yr (>400 μg L-1; 1975 Keller et al. 2002). Two rivers (Aa-Bach and Möchaltdorfer-Aa) flow into the lake, while one lake outlet

- 38 - Chapter 3 Sexual reproduction

(Glatt) drains the system. Only the larger Aa-Bach seems to create a noticeable flow-through toward the lake outlet (Keller et al. 2002). Daphnia galeata, D. hyalina and their interspecific hybrids occur in Greifensee (Spaak et al. 2000a).

Field methods – Quantitative Daphnia samples were collected weekly (every other week during winter) beginning in February 1999 with a 95-μm net and preserved in 95% ethanol. Samples were taken from three distinct stations (all in the deepest part of the lake), to prevent the sampling of zooplankton patches. Adult asexual females without parthenogenetic eggs, adult females with parthenogenetic eggs, juveniles, ephippial females, and ephippia were enumerated separately, to calculate Daphnia densities in the lake. Qualitative Daphnia samples were taken with a 250-μm plankton net, to collect adult females for genetic analyzes. Sexual females (arrows in Fig. 1A) and males (if present) also were collected, to get an adequate number of individuals for genetic analyzes. Samples were stored in liquid nitrogen for later analyzes. Floating ephippia, just released into the water column from sexual females, were qualitatively sampled by carefully skimming wide areas of the water surface with a 100-μm plankton net. Ephippia were stored in the dark at 4qC (155-783 d) until they were exposed to hatching stimuli. Sediment cores of Greifensee (1999, 2000) were sampled and sliced into 1.4-cm-thick slabs, and ephippia were removed with forceps and stored in the same way as the floating ephippia. Keller et al. (2002) provides further details about the sediment cores.

Ephippia hatching – After the cold incubation in the refrigerator, all ephippia from the sediment cores and subsamples from floating ephippia (numbers ranged from 398 in May 2001 to 1,530 in August 2001) were counted and placed in six-well culture plates filled with filtered (0.45 μm) lake water and exposed to a long-day photoperiod (16 h light : 8 h dark) at 12qC, to break diapause (Weider et al. 1997). Ephippia were monitored daily for 4 weeks and thereafter every second day for another 4 weeks. Hatched daphnids were transferred to separate culture jars (50 ml), and individually raised as clonal cultures.

Hatching success – Damaged ephippia (broken in two parts), intact ephippia, and viable ephippial eggs (dark green in color) were counted in subsamples using a stereo microscope. Ephippia were opened if eggs were not visible from

- 39 - Sexual reproduction Chapter 3 the outside, and the total number of viable ephippial eggs per sample was calculated as V/M × 100; where V is the number of total viable eggs: the sum of hatchlings and the remaining unhatched viable eggs (olive green in color), and M is the number of maximal possible eggs: number of intact ephippia × 2, because one ephippium can contain in maximum of two diapausing eggs. The percentage of total viable eggs that hatched (hatching success) was calculated as H/V × 100; where H is the number of hatchlings and V is the number of total viable eggs (see above).

Table 1. Possible genotype classes after two generations of hybridization with two species- specific markers and the genealogical class interpretation based on the method of Nason and Ellstrand (1993).

Genotype Genealogical class interpretation

AABb *BPgaleata, F2

AaBB *BPgaleata, F2

Aabb *BPhyalina, F2

aaBb *BPhyalina, F2

AaBb *F1, BPgaleata, BPhyalina, F2

AAbb *F2

aaBB *F2

AABB *Pgaleata, BPgaleata, F2

aabb *Phyalina, BPhyalina, F2 * Genealogical class to which each of the nine possible genotype categories was classified. BP: backcrossed genotype, P: parental genotype

Allozyme electrophoresis and statistical analyzes – Four polymorphic enzyme loci were screened for asexual females, sexual females, males, and ex- ephippial hatchlings using cellulose acetate electrophoresis: aldehyde oxidase (AO; enzyme commission number [EC] 1.2.3.1.), aspartate amino transferase (AAT; EC 2.6.1.1), phosphoglucose isomerase (EC 5.3.1.9), and phosphoglucomutase (EC 5.4.2.1). Two of these loci, AAT (Wolf and Mort 1986) and AO (Gießler 1997), have diagnostic alleles for distinguishing between D. galeata and D. hyalina. Using two diagnostic markers, six possible genealogical classes can be identified (for a discussion on characterisation, see Nason and Ellstrand 1993). This raw classification reflects only an incomplete estimate of natural frequencies of genealogical classes. Misclassification, especially in F2 hybrids with nine possible genotypes, is unavoidable (Tab. 1). To reduce misclassification, genealogical class identification was done using

- 40 - Chapter 3 Sexual reproduction the computer program of Anderson and Thompson (2002), which uses Bayesian statistical methods to calculate the probability that an individual belongs to various hybrid categories. Because of the low number of available loci (four), we could only distinguish among one parental species (D. galeata),

F1 hybrids (probability >95%), and all others. These remaining individuals could not be classified and were pooled as ‘backcrosses’ (probability <95%). Thus, the group ‘backcrosses’ is a mixed category. Each individual was analyzed three times with a distinct number of “sweeps” (100,000, 200,000, and 700,000; for a definition of this method, see Anderson and Thompson 2002) to check whether differences in calculated probabilities were <0.01 between the different sweeps (E. C. Anderson pers. comm.). Tests of differentiation were used to determine differences in genetic structure for date, examining sexual and asexual females, asexual, sexual females and males, and asexual, sexual females and hatchlings (Goudet 2001). FSTAT analyzes the overall loci G-statistic to classify tables by randomizing multi locus genotypes between each pair of samples. Correlations between hatching success and numbers of viable eggs in floating ephippia sampled during spring and fall were tested with STATISTICA (Statsoft), to verify the relationship between egg quality and reproductive success. The effect of storage time in the sediment on hatching success also was tested, because Weider et al. (1997) had previously found a decreased hatching success with increasing ephippia age. Cáceres and Tessier (2003) found an increased degeneration of laboratory-stored diapausing eggs; we therefore tested whether storage time in the laboratory was correlated with the number of viable eggs in floating ephippia.

Table 2. Classification of genetically analyzed asexual females (1998–2002), according to Nason and Ellstrand (1993, six genealogical classes) and Anderson and Thompson (2002, three genealogical classes). For further explanation, see the text.

Nason’s Anderson’s classification

classification Pgaleata F1 Backcrosses Total Pgaleata 823 1 183 1,007

F1 0 8,506 14 8,520 BPgaleata 10 962 619 1,591 Phyalina 0 0 48 48 BPhyalina 0 147 468 615

F2 0 0 83 83 Total 833 9,616 1,415 11,864

- 41 - Sexual reproduction Chapter 3

Results Using the approximate six-genotype classification of Nason and Ellstrand

(1993), asexual daphnids (collected during 1998 – 2002) were dominated by F1 hybrids at all dates (~80%; Fig. 1A), except those from April 2002, when the parental species D. galeata (Pgaleata) dominated the population (~70%). Indi- viduals with a backcrossed D. galeata and D. hyalina genotype occasionally occurred in proportions up to 20%, whereas the animals with a F2 hybrid and parental D. hyalina (Phyalina) genotype never made up more than ~5% of the whole population (Fig. 1A). The density fluctuations (500,000 individuals m-2 in May 1998 to 3,000 individuals m-2 in February 2002) did not affect the distribution pattern of genotype classes described above (Fig. 1B). Using the statistically more rigorous three-genotype classification of Anderson and Thompson (2002), we observed a pattern comparable to that of the six- genotype classification (Fig. 1A, C). The model of Anderson and Thompson classified 1,096 more individuals as F1 hybrids and 174 fewer individuals as

Pgaleata (Tab. 2). All animals with a Phyalina and F2 hybrid genotype could not be classified by the Anderson and Thompson model and were added to the backcrossed group. Periods during which sexual females were present in Greifensee (arrows in Fig. 1A) varied from 1 week to 1 month (B. K., pers. observation). Sexual reproductive periods could be determined during spring and fall by the presence of sexual females, males, and newly produced floating ephippia (Figs. 1A, 2). Males occurred in the absence of sexual females on several occasions (Fig. 2). The viable egg proportion of floating ephippia varied from 21% (May 2000) to 2% (August 2001; Fig. 2). The proportion of viable eggs in floating ephippia and the hatching success was highly correlated (r2 = 0.883, P = 0.005). Most hatching events occurred in May 2000 (62% of all viable eggs) and April 2002 (67%). The storage time of floating ephippia in our refrigerator ranged from 155 d (April 2002) to 738 d (October 2000) and was not correlated with hatching success (r2 = 0.017, P = 0.81). We compared the sexual and asexual females for the six dates on which they were sampled simultaneously using the three genealogical classes of Anderson and Thompson (2002) (Fig. 3). The genetic and genealogical class composition differed significantly between sexual and asexual females on all dates except during spring 2002 (Fig. 3). Pgaleata tended to be more abundant in the sexual females (five of six samples). No clear pattern was observed for

F1 hybrids. They occurred in higher proportion in asexual females on four

- 42 - Chapter 3 Sexual reproduction dates (spring 1998 and 2000 and autumn 2000 and 2001), but, on the two remaining dates, the distribution was opposite. In October 2000, we collected a sufficient number of males (n = 20) to include them in our genetic analysis. Comparing males with asexual and sexual females, the most obvious difference was the complete absence of Pgaleata in the group of males and asexual Daphnia, whereas its proportion was >25% in the sexual females (Fig. 4). F1 hybrids dominated all reproductive stages – 94% in the asexual females, 80% in the males, and 66% in the sexual females (Fig. 4).

700 100 empty 600 ephippia 80 viable eggs ) failed to hatch

-3 500 hatchlings 60 400 died hatchlings 300 40 survived 200 males individuals (m 20 ephippial 100 females

0 0 proportion ephippal of eggs (%) Jun 02 Jun 02 Jun 01 Jun 01 Jun 00 Jun 00 Oct 01 Oct 00 Feb 02 Feb 01 Apr 02 Apr 02 Apr 01 Apr 01 Apr 00 Apr 00 Dec 01 Dec 00 Aug 01 Aug 00

Figure 2. Abundance of sexual females and males in Greifensee (April 2000–July 2002) and proportion of ephippial eggs from floating ephippia (successful hatchlings, hatchlings died after hatching, viable ephippial eggs that did not hatch, and empty ephippia). Empty ephippia were used to calculate the maximal number of sexual eggs (2 × the number of intact ephippia). Viable ephippial eggs were calculated as the sum of hatchlings of floating ephippia and unhatched, olive green, diapausing eggs.

Hatchlings of floating ephippia that survived to maturity exhibited a different genealogical class distribution than the asexual and sexual pelagic females for the dates when ephippia were collected (May 2000 and April 2002). Pgaleata dominated the hatchlings from floating ephippia (75% in 2000 and 71% in 2002), compared with the asexual and sexual females (Fig. 5). The oldest ephippial egg that hatched from the sediment was ~40 yr old. The proportion of viable eggs varied from 23% to 7% (Fig. 6B). Hatching success was correlated weakly with sediment depth (r2 = 0.42, P = 0.043). Pgaleata and the backcrosses dominated the sediment hatchlings (Fig. 6A), as was the case with hatchlings from recently produced ephippia (Fig. 5). We

- 43 - Sexual reproduction Chapter 3

could not identify F1 hybrid genotypes with a probability >95%, but, most likely, they are hidden in the backcrossed group.

350 113 51 106115 83 100 asexual females 80 Pgaleata F1 60 backcrosses

0 16 96 32 75 61 174 100

proportion (%) sexual females 80 *****NS 60

0 Jan 02 Jan Jan 98 Jan 99 Jan 00 Jan 01 Jan Sep 02 Sep 98 Sep 99 Sep 00 Sep 01 May 00 May 01 May 02 May 99 May 98

Figure 3. Proportion of three genealogical classes (Anderson and Thompson 2002) in the asexual (top) and sexual females (bottom) of a hybridizing Daphnia complex in Greifensee. The top of each bar indicates the number of individuals analyzed. Significant differences were based on the pairwise test of differentiation between sample pairs (FSTAT version 2.9.3.1; Goudet 2001) *Significant differences after Bonferroni correction (1,000 permutations, P = 0.05). NS: not significant.

Discussion Our data show that the genetic composition of sexual females and males, as well as hatchlings from both recent (pelagic) and old (sediment) ephippia, is significantly different from the genetic composition of pelagic asexual

Daphnia. Of interest, the F1 hybrids dominate the pelagic population (Fig. 1A) but are nearly absent in the recently produced ephippia, as well as in the ephippia found in the diapausing egg bank. This finding is remarkable, because

- 44 - Chapter 3 Sexual reproduction sexual hybrids were found frequently in the pelagic population (Figs. 3 and 4), which indicates they should be capable of producing ephippia with the hybrid genotype. The genetic pattern of daphnids hatched from recent ephippia is comparable to those hatched from the sediment. Thus, we presume that comparable processes may have led to these similar genetic compositions over time. The high hatching success of floating ephippial eggs (Fig. 2) and the dominance of D. galeata in hatchlings (Fig. 5) leads us to speculate that the remaining unhatched viable eggs also belong to D. galeata and most probably not to F1 hybrids.

Individuals with a F1 hybrid genotype dominated the asexual females in Greifensee over the past 4 yr, and backcrossed hybrids (mainly backcrossed D. galeata genotypes) were represent at a rate of 10-20% throughout the study period (Fig. 1A). This is in contrast to Lake Constance (Jankowski 2002), in which the same hybridizing taxa co-occur. Jankowski (2002) found no hybrid dominance and a much higher proportion of backcrossed D. hyalina using the same classification method of Nason and Ellstrand (1993) that we used. These contradictory findings suggest an opposite introgression pattern of alleles within the two species in Greifensee and Lake Constance. Spaak et al. (2000a) suggested that, in Greifensee, pure D. hyalina probably never have been present, which might explain this dissimilar pattern. Another reason may be that the two lakes exhibit differences in environmental conditions that may favor different genealogical classes. Taxonomic differences can be influenced by the ability of different genealogical classes to cope with alteration in food composition (Repka et al. 1999), fish predation (Spaak et al. 2000b), or parasites (Wolinska et al. 2004). 77 32 20 100

80 Pgaleata 60 F1 40 backcrosses 20 proportion (%) 0 asexual sexual males females females Figure 4. Proportion of three genealogical classes (Anderson and Thompson 2002) in the sexual females, asexual females, and males of the hybridizing Daphnia species complex in Greifensee, October 2000. The top of each bar indicates the number of analyzed individuals. All groups are significantly different from each other (after Bonferroni correction; 3,000 permutations, P < 0.017; pairwise test of differentiation between sample pairs; FSTAT version 2.9.3.1; Goudet 2001).

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Genealogical class analyzes of closely related hybridizing and backcrossing species, as in the Daphnia galeata × hyalina complex, without clearly morphological distinguishable characters and only a limited number of diagnostic genetic markers, can lead to misclassification problems. Most allozyme studies on hybridizing Daphnia populations have been based on one (e.g., Wolf and Mort 1986; Weider et al. 1997) or two (e.g., Jankowski 2002) diagnostic markers. The comparison of the classification models from Nason and Ellstrand (1993) and Anderson and Thompson (2002) has shown that the overall taxa pattern does not change (Fig. 1A,C), although several individuals were classified differently. But the number of available loci (four) we used in computing the Anderson and Thompson (2002) model is too low to distinguish more detailed patterns; therefore all D. hyalina and F2 hybrids were lumped together in the backcrossed group (Table 2).

May 2000 Apr 2002 123 96 64 102 137 69 100 Figure 5. Proportion of three different 80 genealogical classes (Anderson and Thompson 2002) in the sexual, asexual females, and exephippial 60 Pgaleata hatchlings of a hybridizing Daphnia F 1 complex in Greifensee on May 2000 40 backcrosses and April 2002. The top of each bar

proportion (%) indicates the number of analyzed 20 individuals. All groups are significantly different from each other 0 in both year (after Bonferroni correction; 3,000 permutations, P < 0.017; pairwise test of differentiation between sample pairs; FSTAT

hatchlings hatchlings version 2.9.3.1; Goudet 2001), except asexual and sexual females in April sexual females sexual females sexual asexual females asexual females asexual 2002.

We found differences in the genealogical class composition between asexual and sexual generations, as was also reported by Jankowski (2002). D. galeata was significantly more abundant in the sexual females, which suggests a higher level of “sexual activity” for D. galeata. Jankowski (2002) found a high proportion of sexual D. galeata (90%) but no sexual D. hyalina during early summer in Lake Constance. In autumn, the pattern was reversed. Hybrids reproduced sexually during both seasons, but in low proportions. Such seasonal alteration can result in the reproductive isolation of both parental

- 46 - Chapter 3 Sexual reproduction groups, as Spaak (1995) described in Tjeukemeer (The Netherlands). In Greifensee, we did not observe a genealogical class switch of sexual daphnids between spring and autumn. Additionally, we found much higher proportions of sexual hybrids in Greifensee. The discrepancy in taxa composition of ephippial females and floating ephippial hatchlings (Fig. 5) could be explained by premating intolerances (e.g., assortative or failed mating or the asynchronous presence of males and sexual females) or by postmating intolerances (e.g., cytological fertilization difficulties, discriminating embryo degeneration, selective hatching, and biased juvenile survival rate). The genealogical class differences between asexual and sexual generations (Figs. 3, 4), in combination with the low number of individuals engaging in sexual reproduction (Fig. 2), suggest non-random (assortative) clonal participation in sexual reproduction. Assortative mating can not completely explain the near absence of hybrids in the pelagic and sediment hatchlings (Figs. 5, 6), because a large portion of F1 hybrids were present in both sexual females and males (Figs. 3, 4). The presence of hybrids and backcrosses in Greifensee indicate that hybrids are able to hatch. The large proportion of empty floating ephippia (Fig. 2) is difficult to explain. The temporal separation of males and sexual females may be important (Fig. 2), but failed mating can not be excluded. Biased cytological fertilization difficulties or discriminating embryo degeneration may also be important factors, but we have very little knowledge of this for Daphnia. Boersma and Vijverberg (1995) found no difference in egg mortality between D. galeata and D. galeata × cucullata hybrids in parthenogenetic eggs, but Schwenk et al. (2001) described failed artificial reciprocal crosses between D. galeata and D. cucullata. If sexual females are fertilized and diapausing eggs are successfully produced, clonal or species-specific selective hatching or biased juvenile mortality might occur. Schwenk et al. (2001) found that hatching and juvenile survival rates of artificially produced D. galeata × cucullata hybrids were relatively low compared with parental species. Because we applied only one set of hatching stimuli (12qC and a long-day photoperiod), we cannot exclude the loss of some hatchlings that might not have been able to hatch under these conditions. The high hatching success (>60% on May 2000 and April 2002; Fig. 2) and the significant positive correlation (r2 = 0.883) between hatching success and the number of viable eggs suggests that this effect might not be strong. Moreover Cáceres and Tessier (2003) described an increased senescence rate of unhatched sexual eggs accompanied by increased hatching success under laboratory conditions compared to natural conditions. We did

- 47 - Sexual reproduction Chapter 3 not observe increased degeneration in our laboratory-stored floating ephippia, as indicated by the significant correlation of the percentage of viable eggs with hatching success (r2 = 0.883, P = 0.005) and a lack of correlation between storage period and viability of eggs (r2 = 0.017, P = 0.81). However, we cannot exclude processes that might affect species and clones differently and thus influence the genealogical class composition of hatchlings. Finally, the low proportion of F1 hybrid hatchlings compared with the sexual F1 hybrid females (Fig. 5) strongly suggests the presence of pre- or postmating barriers. This is also supported by the low proportion of viable eggs that we found in the floating ephippia, which were produced when mainly hybrid sexual females were abundant in the lake (Figs. 2, 3, 4).

number of hatchlings

0 102030405060 0 A 2.1 2 A 6.3 4 10.0 14.0 6 19.2 8 24.8 30.4 Figure 6. (A) Genealogical class 10 36.4 distribution (Anderson and sediment depth (cm) sediment 12 backcrosses 42.3 age (years) sediment Thompson 2002) and abun- Pgaleata 49.0 dance of ex-ephippial hatchlings 14 0 in the sediment of a hybridizing B 2.1 Daphnia complex in Greifensee 2 hatched eggs B 6.3 viable eggs (total number of matured 4 empty ephippia 10.0 neonates). (B) The distribution 14.0 of ephippial eggs (successful 6 19.2 hatchlings, hatchlings that died 8 24.8 after hatching, viable ephippial 30.4 eggs that did not hatch, and 10 36.4 empty ephippia). Empty ephip- sediment depth(cm) sediment 12 42.3 age (years) sediment 49.0 pia are equated to maximal 14 possible eggs, which were cal- 0 50 100 150 200 250 300 culated as 2 × the number of 3 -2 intact ephippia. number of eggs (10 x m )

In our study, genotype class patterns drawn from floating ephippia hatchlings (Fig. 5) were comparable with those from the sediment (Fig. 6A), and both differed from the asexual pelagic females (Fig. 1C). Similar to our findings, Weider et al. (1997) found large differences between the asexual pelagic daphnids and ex-ephippial hatchlings from the diapausing egg bank in Lake Constance. No D. hyalina and only a few hybrid individuals hatched

- 48 - Chapter 3 Sexual reproduction from sediment layers dating from years during which those taxa were present in the lake (Weider and Stich 1992). Weider et al. (1997) suggested, on the basis of high hatching success (up to 100% of the viable eggs) and analogous allele frequencies in hatchlings and corresponding pelagic population, that hatchlings are a random subsample of the diapausing egg pool. They hypothesized that differences in taxa composition may result from taxon- specific buoyancy or the inability of D. hyalina to produce viable diapausing eggs. Jankowski (2002) found shifts in various taxa hatched from sediments in littoral and deep water. This suggests taxon-specific buoyancy of ephippia. In

Greifensee, the low frequency of recent F1 hybrid hatchlings (floating ephippia) and the simultaneous presence of sexual F1 hybrids (Fig. 5), compared with the absence of hybrids in sediment hatchlings (Fig. 6A), suggest that very few viable hybrid ephippia were produced in the past. However, we cannot conclude from our data that hybrids did not occur in the lake during this time.

Our findings – the F1 hybrid dominance in the pelagic asexual females, the low abundance of sexual individuals, the genealogical class differences between asexual, sexual generations, and ephippial hatchlings and the ephippia quality dissimilarities – strongly suggest that clonal reproduction is the more efficient short-term strategy for the Daphnia hybrid swarm in Greifensee. Clonal reproduction is characterized by its inflexibility to react to changes in environmental conditions, through a lack of recombination. Clonal reproduction also risks the accumulation of deleterious mutations (e.g. Palsson 2001) or easier adaptations of parasites (Hamilton 1980; Hurst and Peck 1996). Our results indicate that selection seems to occur against sexually reproducing hybrids. This may explain why Pgaleata and hybrid taxa stay distinct and co- occur in Greifensee. In addition, our results suggest that the Daphnia diapausing egg bank may not represent the past population in the lake. No statement can be made about the former taxon distribution of hybrid swarms analyzing diapausing egg banks; our results show that the absence of taxa in the sediment does not indicate their absence in past populations. The low percentage of hatchlings in several sediment layers must therefore be explained by the presence of high numbers of degenerated hybrid-diapausing eggs, whereas ephippia from time horizons in the sediment with high hatching success may have been produced by D. galeata.

- 49 - Sexual reproduction Chapter 3

Acknowledgements We thank Christine Dambone-Boesch for help in the laboratory; Regula Illi for counting quantitative samples; and Esther Keller, Hans Ruedi Bürgi, and Richard Illi for support in the field. We thank Eric C. Anderson for his help and advice concerning his software and help interpreting the data. We also thank Sandra Lass, Mike Monaghan, Nelson Hairston, Jr., and two anonymous reviewers for useful comments on an earlier version of the manuscript. P. S. thanks Charles Kerfoot for the invitation to attend the Biocomplexity symposium in Ann Arbor. This work was supported by grant 31-65003.01 from the Swiss National Science Foundation to P.S.

References Anderson, E. C., and E. A. Thompson. 2002. A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160: 1217-1229. Boersma, M., and J. Vijverberg. 1995. The significance of nonviable eggs for Daphnia population dynamics. Limnol. Oceanogr. 40: 1215-1224. Branvall, M. L., R. Bindler, O. Emteryd, and I. Renberg. 2001. Four thousand years of atmospheric lead pollution in northern Europe: a summary from Swedish lake sediments. J. Paleolimn. 25: 421-435. Cáceres, C. E. 1998. Interspecific variation in the abundance, production, and emergence of Daphnia diapausing eggs. Ecology 79: 1699-1710. Cáceres, C. E., and A. J. Tessier. 2003. How long to rest: the ecology of optimal dormancy and environmental constraint. Ecology 84: 1189-1198. Campton, D. E., and F. M. Utter. 1985. Natural hybridization between steelhead trout (Salmo gairdneri) and coastal cutthroat trout (Salmo clarki clarki) in 2 Puget Sound Streams. Can. J. Fish. Aquat. Sci. 42: 110-119. Cousyn, C., L. De Meester, J. K. Colbourne, L. Brendonck, D. Verschuren, and F. Volckaert. 2001. Rapid local adaptation of zooplankton behavior to changes in predation pressure in absence of neutral genetic changes. Proc. Natl. Acad. Sci. U. S. A. 98: 6256-6260. Duffy, M. A., L. J. Perry, C. M. Kearns, L. J. Weider, and N. G. Hairston. 2000. Paleogenetic evidence for a past invasion of Onondaga Lake, New York, by exotic Daphnia curvirostris using mtDNA from dormant eggs. Limnol. Oceanogr. 45: 1409-1414.

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Duigan, C. A., and H. H. Birks. 2000. The late-glacial and early-Holocene palaeoecology of cladoceran microfossil assemblages at Krakenes, western Norway, with a quantitative reconstruction of temperature changes. J. Paleolimn. 23: 67-76. Frey, D. G. 1974. Paleolimnology. Mitt. Internat. Verein. Limnol. 20: 95-123. Gießler, S. 1997. Analysis of reticulate relationships within the Daphnia longispina species complex. Allozyme phenotype and morphology. J. Evol. Biol. 10: 87-105. Gomez, A., and G. R. Carvalho. 2000. Sex, parthenogenesis and genetic structure of rotifers: microsatellite analysis of contemporary and resting egg bank populations. Mol. Ecol. 9: 203-214. Goudet, J. 2001. FSTAT (version 2.9.3.1), a program for IBM PC compatibles to calculate Weir and Cockerham's (1984) estimators of F-statistics. Institut de Zoologie et Ecologie Animale, Université Lausanne. Hairston, N. G. and others 2001. Natural selection for grazer resistance to toxic cyanobacteria: Evolution of phenotypic plasticity? Evolution 55: 2203-2214. Hairston, N. G., L. J. Perry, A. J. Bohonak, M. Q. Fellows, C. M. Kearns, and D. R. Engstrom. 1999. Population biology of a failed invasion: Paleolimnology of Daphnia exilis in upstate New York. Limnol. Oceanogr. 44: 477-486. Hamilton, W. D. 1980. Sex versus non-sex versus parasite. Oikos 35: 282–290. Hofmann, W. 1991. The late-glacial/holocene Bosmina (Eubosmina) fauna of Lake Constance (Untersee) (F.R.G.): traces of introgressive hybridization. Hydrobiologia 225: 81-85. Hurst, L. D., and J. R. Peck. 1996. Recent advances in understanding of the evolution and maintenance of sex. Trends Ecol. Evol. 11: A46–A52. Jankowski, T. 2002. From diapause to sexual reproduction: Evolutionary ecology of the Daphnia hybrid complex from Lake Constance. Ph. D. University of Konstanz. Keller, B., H. R. Bürgi, M. Sturm, and P. Spaak. 2002. Ephippia and Daphnia abundances under changing throphic conditions. Verh. Internat. Verein. Limnol. 28: 851-855. Kerfoot, W. C., J. A. Robbins, and L. J. Weider. 1999. A new approach to historical reconstruction: Combining descriptive and experimental paleolimnology. Limnol. Oceanogr. 44: 1232-1247. Limburg, P. A., and L. J. Weider. 2002. “Ancient” DNA in a microcrustacean resting egg bank can serve as a paleolimnological database. Proceedings of the Royal Society of London Series B- Biological Sciences 269: 281-287. Lotter, A. F., H. J. B. Birks, W. Hofmann, and A. Marchetto. 1998. Modern diatom, cladocera, chironomid, and chrysophyte cyst assemblages as quantitative indicators for the reconstruction of past environmental conditions in the Alps. II. Nutrients. J. Paleolimn. 19: 443-463.

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Nason, J. D., and N. C. Ellstrand. 1993. Estimating the frequencies of genetically distinct classes of individuals in hybridized populations. Journal of Heredity 84: 1-12. Palsson, S. 2001. The effects of deleterious mutations in cyclically parthenogenetic organisms. Journal of Theoretical Biology 208: 201-214. Reid, V. A., G. R. Carvalho, and D. G. George. 2000. Molecular genetic analysis of Daphnia in the English Lake District: species identity, hybridisation and resting egg banks. Freshwat. Biol. 44: 247-253. Repka, S., S. Vesela, A. Weber, and K. Schwenk. 1999. Plasticity in filtering screens of Daphnia cucullata x galeata hybrids and parental species at two food concentrations. Oecologia 120: 485-491. Schwartz, S. S., and P. D. N. Hebert. 1987. Methods for activation of the resting eggs of Daphnia. Freshwat. Biol. 17: 373-379. Schwenk, K., M. Bijl, and S. B. J. Menken. 2001. Experimental interspecific hybridization in Daphnia. Hydrobiologia 442: 67-73. Schwenk, K., and P. Spaak. 1995. Evolutionary and ecological consequences of interspecific hybridization in cladocerans. Experientia 51: 465-481. Spaak, P. 1995. Sexual reproduction in Daphnia: interspecific differences in a hybrid species complex. Oecologia 104: 501-507. Spaak, P. 1996. Temporal changes in the genetic structure of the Daphnia species complex in Tjeukemeer, with evidence for backcrossing. Heredity 76: 539-548. Spaak, P. 1997. Hybridization in the Daphnia galeata complex: are hybrids locally produced? Hydrobiologia 360: 127-133. Spaak, P., L. Eggenschwiler, and H. Bürgi. 2000a. Genetic variation and clonal differentiation in the Daphnia population of the Greifensee, a pre-alpine Swiss lake. Verh. Internat. Verein. Limnol. 27: 1919-1923. Spaak, P., J. Vanoverbeke, and M. Boersma. 2000b. Predator induced life history changes and the coexistence of five taxa in a Daphnia species complex. Oikos 89: 164-174. Statsoft, I. 2001. STATISTICA (data analysis software system), version 6. www.statsoft.com. Weider, L. J., W. Lampert, M. Wessels, J. K. Colbourne, and P. Limburg. 1997. Long-term genetic shifts in a microcrustacean egg bank associated with anthropogenic changes in the Lake Constance ecosystem. Proceedings of the Royal Society of London Series B- Biological Sciences 264: 1613-1618. Weider, L. J., and H. B. Stich. 1992. Spatial and temporal heterogeneity of Daphnia in Lake Constance; intra- and interspecific comparisons. Limnol. Oceanogr. 37: 1327-1334. Wolf, H. G., and M. A. Mort. 1986. Interspecific hybridization underlies phenotypic variability in Daphnia populations. Oecologia 68: 507-511.

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Wolinska, J., B. Keller, K. Bittner, S. Lass, and P. Spaak. 2004. Do parasites lower Daphnia hybrid fitness? Limnol. Oceanogr. 49: 1401-1407.

- 53 -

CHAPTER 4

Pre- and postzygotic reproductive isolation keep hybridizing Daphnia species distinct with J. Wolinska, C. Tellenbach and P. Spaak Limnology and Oceanography, in press

Abstract Hybridization and introgression are sources of variation that can influence adaptation and speciation. Hybridization might be especially important in species, such as Daphnia, which are cyclical parthenogens that can propagate both asexually and sexually. Daphnia can show hybrid superiority, but the question remains why hybrids do not outcompete parental taxa? One potential explanation for this is reproductive isolation, which in Daphnia could arise at various stages during development. Here we focussed on the question of whether pre - (e.g., assortative mating, temporal isolation) or postzygotic (e.g., hybrid inviability or infertility) barriers are more likely to affect the hybridization between D. galeata and D. hyalina. In hybridizing Daphnia little is known about the effective extent of hybridization due to the low number of diagnostic markers and limited information about sexual reproduction. We analyzed occurrence and taxonomic composition of reproductive stages in the life cycle of D. galeata, D. hyalina and their hybrids in Greifensee (Swit- zerland) using molecular genetic methods. We found evidence for reproductive isolation between the taxa, and that hybrids dominate the asexual Daphnia population but have reduced sexual fitness, providing one of the potential mechanisms for parental taxa to remain distinct. Low sexual fitness limits the hybrids’ abilities to take advantage of diapausing eggs; for instance dispersal ability including colonization of new habitats, survival probability during harsh environmental conditions, heritability of genes, long-term persistence, and might furthermore lead to underestimations of historical hybrid occurrences based on diapausing egg bank reconstructions.

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Introduction Hybridization and introgression are a source of variation that can influence adaptation to new environments and influence speciation in both plant and animal systems (e.g., Anderson and Stebbins 1954). Hybridization is a common phenomenon in both aquatic and terrestrial habitats (reviewed in Dowling and Secor 1997), but seems to be more common among plant (25% species hybridize) than animal species (at least 10%, reviewed in Mallet 2005). It occurs after secondary contact between partially reproductively isolated species, and has several potential outcomes. Hybridization can lead to extinction of one or both species (reviewed in Rhymer and Simberloff 1996), to coexistence of parental species and hybrids (Moore 1977), to reinforcement (reviewed in Butlin 1987), or to merging into novel populations of reticulate or polyphyletic origin due to introgressive hybridization (reviewed in Arnold 1992). The evolutionary significance of hybridization depends strongly on the frequency of hybridization events as well as on hybrid fitness (reviewed in Arnold 1992). Both factors hinge on reproductive isolation mechanisms that can be found in various numbers, combinations and strengths between the species involved (e.g., Arnold 1997), and be either pre- or postzygotic (reviewed in Avise 1994). Prezygotic isolation mechanisms include cytonuclear incompatibility and a range of behavioural and mechanical mating barriers (e.g., viability selection on migrants, assortative mating, egg-sperm recognition, and timing of reproduction). Postzygotic incompatibilities, on the other hand, comprise reduced hybrid fitness (e.g., offspring viability, fertility) as a consequence of genomic divergence and thus result in incompatibilities. Interactions between parental genomes can lead to a variety of positive and negative epistatic effects on the nuclear and cytonuclear level and result in a set of different genotypes of varying fitness (Burke et al. 1998). However, the final success of hybrids in a specific habitat depends on a complex genotype- by-environment interaction (e.g., Campbell and Waser 2001) as well as on stochastic effects related to extinction and colonization (Babik et al. 2003). Although in general hybrids seem to perform poorly compared to their parental species (e.g., Arnold 1997), under specific environmental conditions they can be equally fit or more fit than their parents (e.g., Burke et al. 1998), which in turn can result in hybrid dominance (e.g., Milne et al. 2003). Hybridization might be especially important among plant and animal species that can propagate both sexually and asexually. Even if F1 hybrid genotypes have reduced sexual fertility, they may be able to perpetuate over years

- 56 - Chapter 4 Reproductive isolation via asexual reproduction and reach high abundances. This should then increase the chance for successful sexual reproduction. In this way, asexual reproduction can facilitate the production of later hybrid generations (e.g., F2 hybrids and backcrosses), self-fertilization, and finally, introgression into the parental population. For example, these mechanisms have been demonstrated in hybrid Iris populations (Burke et al. 2000). Hybridization is especially common among zooplankton species, even across different genera of rotifers and cladocerans (reviewed in Hebert 1985), which reproduce by cyclical parthenogenesis. In cyclic parthenogens, long asexual phases are interrupted by short periods of environmentally-induced sexual reproduction. In cladocerans, for example, females switch to the production of males and sexual haploid ephippial eggs. Fertilised eggs are released in a protecttive structure called an ephippium, and hatch after a period of diapause when exposed to hatching stimuli. Ephippia are important for colonizing new habitats and re-establishing local populations after unfavourable conditions. They can stay viable for decades (e.g., Carvalho and Wolf 1989). The most frequently studied cyclic parthenogens in aquatic systems are cladocerans of the genus Daphnia. Hybrids between D. galeata, D. cucullata, and D. hyalina are commonly found sympatrically with their parental species (D. galeata-hyalina-cucullata species complex) in permanent lakes across Europe. This co-occurrence of taxa can be explained by taxon-specific fitness differences indicated by distinct niche breadths (Weider 1993). Accordingly, fluctuations in biotic and abiotic conditions modify taxon-specific selection pressures and alter taxonomic composition. However, hybrids often combine life-history traits of their parents in a beneficial way (e.g., Schwenk and Spaak

1995), leading to F1 hybrid dominance (e.g., Schwenk and Spaak 1995; Keller and Spaak 2004), also known as “temporal hybrid superiority” (proposed by Spaak and Hoekstra 1995). Long-term co-existence of parental species and hybrids (e.g., Keller and Spaak 2004) is only possible under conditions that also favour parental taxa and strengthen their competitive ability. For example, the parental taxon can be more resistant to a harmful parasite than its F1 hybrid offspring (Wolinska et al. 2004). High hybrid competitive abilities have previously been discussed (e.g., Spaak and Hoekstra 1995), but the question remains why taxa do not merge? One possible explanation is the presence of reproductive isolation mechanisms that prevent parental species and hybrids from merging. Although observed differences in taxonomic composition between hatchlings from ephippial (sexual) eggs and active (asexual) lake

- 57 - Reproductive isolation Chapter 4 populations (e.g., Keller and Spaak 2004) suggest the existence of reproductive isolation, these studies did not identify at which stage of the sexual Daphnia life cycle this bias occurs. There are many stages within the sexual Daphnia reproduction cycle that could potentially be influenced by reproductive isolation mechanisms and lead to a shift in taxonomic composition between generations. These stages are (Fig. 1): ‘transition from asexual to sexual morphs’ (induction of males and females, which produce gametes), ‘mating’ (temporal and spatial co- occurrence of males and sexual females and their mating success), ‘ephippial (sexual) egg development’ and ‘hatching’. With‘transition from asexual to sexual morphs’, signifycant taxon shifts have been reported in the production of both sexual females and males (e.g., Spaak et al. 2004). Seasonal differences in the timing of the production of males and sexual females (‘mating’) were found among parental Daphnia taxa: D. galeata usually reproduce sexually in spring (e.g., Jankowski 2002), whereas D. hyalina and D. cucullata do so in fall (e.g., Spaak 1995). In some populations, however, sexual individuals of different parental taxa co-occur, enabling hybridization (e.g., Spaak et al. 2004). In addition to temporal separation, Daphnia taxa (e.g., Stich and Lampert 1981) or even genders within the same taxon (Brewer 1998) can have different depth preferences in the water column. In the D. galeata- hyalina-cucullata species complex, however, no taxon or gender specific depth distribution has been found (Spaak et al. 2004). In a crossing experiment between two D. galeata and two D. cucullata clones, the presence of mating barriers was manifested in reduced mating success and low hatching rates of interspecific crosses (i.e., ‘ephippial egg development’ and ‘hatching’, Schwenk et al. 2001). To date, however, the entire sexual reproducetive cycle has not been studied for any natural hybridizing Daphnia population. The aim of our study was to analyze the reproductive cycle in the D. galeata-hyalina-cucullata species complex and to examine at which stage reproductive isolation mechanisms arise. Life history traits explaining hybrid fitness and maintenance in asexual lake populations have been described in detail (e.g., Schwenk and Spaak 1995), but the processes controlling the genetic structure of hybridizing populations (e.g., the relative strengths of pre- and postzygotic barriers) are not well understood. Here, we focussed on the question of whether pre- or postzygotic barriers affect potential hybridization (Fig. 1; Table 1). Further, we tested the hypothesis that hybrids have reduced sexual fitness compared with parental taxa, which are traded-off by increased asexual reproduction.

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Table 1. Summary of the aspects of the sexual life cycle of hybridizing Daphnia analyzed in order to detect the presence and strength of pre- and postmating reproductive isolation mechanisms.

Analyzed Questions Response variables Predictions stages ‘transition Do parental taxa Frequency of pure parental In absence of reproductive from produce relatively taxa in asexual and sexual isolation the asexual population asexual to more sexual females (spring 2000, 2002, should participate randomly in sexual females? 2004, and 2005). (including sexual reproduction and morphs’ data from Keller and Spaak therefore clonal and taxonomic 2004). composition should be the same Do parental taxa Frequency of pure parental between asexual and sexual produce relatively taxa in asexual females and stages more males? males (fall 2003, 2004, and spring 2004, 2005). ‘mating’ Do sexual females Vertical depth distribution Both sexes should show and males co-occur of males and sexual females overlapping depth distribution to spatially? at noon and midnight. enable mating. ‘egg Are ephippia of Number of pure parental In absence of reproductive develop- hybrids more often and hybrid sexual females isolation hybrids and parental ment’ empty than these with and without ephippial taxa should have equal of parental eggs (pooled data from 32 proportion of empty ephippia. species? checked females sampled in spring 2004 and 59 in 2005, respectively). Are ephippial eggs Number of pure parental Observed taxonomic produced by and hybrid ephippial eggs composition should correspond random mating? (spring 2004, spring 2005); to the one calculated under the Expected hybrid-parental assumption of random mating. distribution - calculated from maternal and paternal taxa composition (see text). ‘hatching’ Does the hybrid- Number of pure parental No difference between parental taxon and hybrids among taxonomic composition of distribution of ephippial eggs and ephippial eggs and hatchlings matured hatchlings hatchlings (spring 2004). would be expected in the correspond with absence of isolation that in ephippial mechanisms. eggs? The first step in Proportion of parental and F1 hybrids, backcrosses and hybridization, i.e., hybrid (F1 hybrids and parental taxa should occur the F1 hybrid backcrossed) hatchlings randomly in matured ex- production, as from floating ephippia (4 yr ephippial hatchlings. shown in other observation period; species, is it also in including data from (Keller Daphnia the most and Spaak 2004). difficult to obtain?

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Material and Methods Study site & field sampling Greifensee (Switzerland) is a eutrophic, prealpine lake of medium size (8.5 km2 surface area; 33 m maximum depth) that has undergone a sharp decline in total phosphorus concentration for the last 25 yr (see Keller et al. 2002). Greifensee is inhabited by Daphnia galeata, D. hyalina and their interspecific hybrids. During months in which sexual reproduction usually occurs (see Keller and Spaak 2004), the Daphnia population was screened weekly for the presence of sexual stages (2004 and 2005). Zooplankton samples were collected with a 250-μm net hauled through the entire water column at three locations around the deepest part of the lake. Samples were cooled with ice during transport to the laboratory and analyzed subsequently. Adult asexual females, sexual females and males were collected for genetic analyses. In spring 2004, ephippia, which were floating on the water surface, were sampled by carefully skimming wide areas of the water surface with a 250-μm plankton net. These ephippia were stored for several months and later exposed to hatching stimuli (i.e. long-day photoperiod with 16 h light and 8 h dark at 12°C, for a more detailed sample description and hatching method see Keller and Spaak 2004). Finally, the overlap in vertical distribution of males and sexual females in the water column was investigated. For this, we used a closing net to sample quantitative 5 meter (30-10 m) and 2.5 m (10-0 m) depth intervals at midday and midnight of two days (28 April and 02 May 2005).

Molecular markers Two sets of genetic markers were used for taxa identification: First, cellulose acetate allozyme electrophoresis was used to analyze four polymorphic enzyme loci (PGI, PGM, AAT, AO, after Keller and Spaak 2004). AO and AAT are diagnostic markers for D. galeata, D. hyalina, and D. cucullata (Wolf and Mort 1986; Gießler 1997). Second, to distinguish diagnostic haplotypes in the D. galeata-hyalina-cucullata species complex, restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer region (ITS) gene region (ITS2, 5.8S, ITS1 and part of 18S) were performed (hereafter referred as ‘ITS-RFLP’). The ITS gene region was amplified following the PCR based procedure of Billiones et al. (2004) and was cut with two restriction enzymes MWO I (NEB; 60qC) and SAU96 (NEB; 37qC). Visualization of fragments was performed on a 2% agarose gel (110-115 volts).

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Sampling procedure and deoxyribonucleic acid (DNA) extraction All individuals were analyzed with allozyme electrophoresis. All sexual females and males (except males in 2004) and a random subsample (20 individuals per sample) of asexual females, were further classified with ‘ITS- RFLP’. For the combined allozyme and RFLP analysis, the following procedure was used: 12 well sample plates (Helena Laboratories) were sterilized for 30 min with bleaching agent (Eau de Javel) and subsequently exposed to UV radiation (>30 min). DNA contamination of the plates was tested by running a PCR with only autoclaved, ultrapure water as template. Individual Daphnia were placed in each well of the plates with 4 μL autoclaved, ultrapure water and homogenized with a spatula. The spatula was sterilized between every homogenization step with a bleaching agent. For DNA preparation, 1.0 μL of each homogenate was incubated for 12 h in 25 μL H3 buffer (10 mmol L-1 Tris-HCl; pH 8.3 at 25°C, 0.05 mol L-1 potassium chloride, 0.005% Tween-20 and 0.005% NP-40) and 1.0 μL Proteinase K (Roche, 18.2 mg mL-1). Proteinase K was deactivated by heating the sample for 12 min to 95°C. PCR and the digesting procedure for ITS-RFLP was performed as described before. For allozyme electrophoresis, 1.0 μL of ultrapure water was added to the remaining Daphnia homogenate and after the first two applications for AO and AAT, 2.0 μL was added, to ensure enough homogenate volume for PGI and PGM (for a more detailed description see Keller and Spaak 2004). Ephippial eggs were analyzed separately from their mothers. Due to the limited amount of cell material, they could only be analyzed for ITS-RFLP. Sexual females were placed on a microscope slide with some drops of autoclaved ultrapure water and ephippia were removed. Ephippia were then placed on a new microscope slide with 100 μL of H3 buffer. The ephippial case was removed and eggs separated from each other. Each egg was squashed and its content transferred to 30 μL H3 buffer for subsequent DNA isolation and ITS- RFLP analysis.

Identification of hybrid classes

Pure D. galeata and F1hybrids (F1 hybrids between D. galeata and D. hyalina) were identified with NewHybrid software (Anderson and Thompson 2002), using four polymorphic allozyme loci (PGI, PGM, AO, AAT). The identify- cation probability was 95%. The remaining individuals (probability <95%) were pooled and labelled as ‘backcrosses’. Furthermore, loci were used to distinguish between different multilocus genotypes (MLG). Clonal diversity D

- 61 - Reproductive isolation Chapter 4 was calculated as the negative logarithm of Simpson’s index of concentration C (seePielou 1975) and is a composite of abundance and evenness (low values indicating a dominance of a single MLG; high values indicating a high number of equally abundant MLGs). Taxon classification using the ITS-RFLP marker was based on the dichotomous key of Petrusek et al. (2005) and was used for direct comparisons among ephippial eggs, asexual females, sexual females, males and hatchlings (except for males in 2004 where only allozyme markers were available). With this method, however, some D. galeata and D. galeata hybrids are incorrectly classified as D. cucullata and D. cucullata hybrids due to a mutation at the restriction site of MWO I (M. Skage, unpublished data). Individuals with no D. cucullata alleles in the two diagnostic allozyme markers (AO, AAT), but with MWO I D. cucullata band pattern, were considered as misclassified. Therefore, homozygous individuals that had a D. cucullata banding pattern and hetero- zygous individuals that had a D. galeata × cucullata banding pattern were classified as D. galeata, and individuals that had a D. cucullata × hyalina banding pattern were classified as D. galeata × hyalina hybrids (M. Skage unpublished data). Analyses with three diagnostic markers (AO, AAT, and ‘ITS-RFLP’) enabled a more precise hybrid classification. Individuals were classified as ‘pure parental’ if they contained exclusively alleles of one taxon at all loci, or otherwise as hybrids. For the taxonomic classification of ephippial eggs, we used ITS-RFLP and our knowledge of their maternal genotypes. An ephippial egg was only classified as ‘pure parental’ if it was ‘pure’ based on ITS-RFLP and if its mother was also classified as a ‘pure parental’ taxon. All other eggs were pooled into the hybrid category.

Analyzes of sexual reproductive cycle in hybridizing Daphnia and statistical analyses The reproductive cycle of Daphnia is divided into the parthenogenetic (clonal) and sexual cycle. In the sexual cycle, selective processes can cause deviations from random mating at several stages (Fig. 1). The different aspects that were analyzed to specify occurrence and strength of pre- and postmating isolation mechanisms are summarized in Table 1. R × C test of independence (Sokal and Rohlf 1995) was used to test for differences in numbers of parental and hybrid sexual females with and without eggs. Further, this test was used to check for taxa differences between numbers of specific reproductive stages at the ‘egg development’-stage (i.e., asexual females, sexual females, males, expected offspring, ephippial eggs and hatchlings; Sokal and Rohlf 1995). At

- 62 - Chapter 4 Reproductive isolation the ‘mating’-stage, we performed a Kolmogorov-Smirnov two sample test to detect differences in clonal diversity between asexual and sexual stages. Furthermore, ANOVA with reproductive stage, reproductive season (spring or fall) and sampling date (nested in reproductive season) as main effects, was used for taxonomic comparison of asexual females and sexual females. For the ANOVA, only those reproductive seasons with at least two samples per season were included. Due to a limited number of seasons with at least two successive samples, we incorporated the single sample from fall 2003 in the analysis and performed a one-tailed paired t-test to compare males and asexual females. Frequency data were arc-sin square-root transformed (Sokal and Rohlf 1995). Assumptions for the calculation of expected taxonomic composition of ephippial eggs were random mating, exclusion of multiple mating events, and that pure offspring can only result from pure parental taxon crosses. Finally, we tested (by analyzing proportions of gravid females) if F1 hybrids have an increased asexual reproduction compared with the parental taxon D. galeata, which might explain their dominance in the asexual population.

asexual reproduction sexual reproduction

growth & physiological 1 3 ‘egg maturation change development‘ ‘transition from 1 partheno- asexual to ‘mating’ genesis sexual morphs’ 5

2 male female development 4 development diapause ‘hatching‘

Figure 1. Reproductive cycle of Daphnia, with critical steps (grey boxes) where reproductive barriers might occur. Numbers refer to the different reproductive stages: (1) asexual female; (2) asexual/male brood (3) sexual female; (4) male, and (5) ephippia with ephippial eggs. (Daphnia drawings from De Meester 1990).

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Results The parental taxa invested significantly more in the production of sexual females than did hybrids (reproductive-stage: F1,6 = 25.45, P = 0.002) but for the male production the tendency was opposite although not significant (t-test: t = 1.67, df = 7, P = 0.069). However, the frequency of the parental taxon (pure D. galeata) in sexual reproduction was variable over the study period, resulting in a significant reproductive-stage × season interaction (F6,6 = 9.16, P = 0.012). Individual clones (MLG-genotypes) also varied in the nature of their participation in sexual reproduction. In particular one clone was almost absent among the sexual females (max. 2.1% in April 2004), whereas it was the dominant clone in males both during autumn 2003 (89.5%) and during spring- summer 2004 (78.8%; Fig. 2). At the ‘mating’-stage we found that the depth distributions of males and sexual females overlapped during both day and night (Fig. 3). At the ‘egg development’-stage 50.0% of the ephippia checked were empty in 2004, and 32.2% in 2005, respectively. We found more empty ephippia produced by hybrid females than by pure parental types (G = 4.134, df = 1, P = 0.042). Furthermore, we detected significantly less hybrid genotypes in the ephippial eggs than would be expected if mating was random (2004: G = 6.623, df = 1, P = 0.010; 2005: G = 52.426, df = 1, P < 0.001; Fig. 4). At the ‘hatching’-stage we found again that less hybrids hatched than would be expected from the genotype distribution of ephippial eggs (2004: G = 4.606, df = 1, P = 0.032). In the 490 hatchlings we analyzed from four consecutive years, only 1.2% were F1 hybrids and 23.3% were D. galeata backcrosses, whereas all the rest were pure D. galeata (based on the NewHybrid software of Anderson and Thompson (2002).

Discussion We found clear evidence for reproductive isolation between taxa within the Daphnia galeata-hyalina-cucullata species complex. Although D. galeata is much less abundant than first-generation D. hyalina × D. galeata hybrids

(F1 hybrids) in the lake, D. galeata dominates the sexual reproduction phase. Reproductive isolation might be the reason why parental species within the D. galeata-hyalina-cucullata species complex are still morphologically and genetically distinguishable even though hybridization among all three taxa is widespread and common.

- 64 - Chapter 4 Reproductive isolation 2003 2004 2005 100 75 A) sexual females 50 25 0 100 75 B) males 50 25

proportion (%) proportion 0 100 75 C) asexual females dominant 'male-MLG' 50 remaining MLG's 25 0 Jan May Sep Jan May Sep Jan May month Figure 2. Temporal fluctuations of the dominant male-multi locus genotype (MLG), (a ‘backcross’ genotype) in (A) sexual females, (B) males, and (C) asexual females in comparison with all other multilocus genotypes (Greifensee; January 2003 - July 2005, samples pooled monthly; sexual stages: only data shown with >20 males or sexual females sampled).

Previous work has found taxonomic shifts among asexual, sexual stages and hatchlings in hybridizing Daphnia (e.g., Jankowski 2002; Keller and Spaak 2004). From these studies it is not clear, however, if the observed differences were the result of pre- or postzygotic mating barriers. The incorporation of kinship relations (i.e. direct comparison of mothers and daughter eggs) helps to disentangle the exact stage at which mating barriers occur. However, at the ‘transition from asexual to sexual morphs’-stage we found directionality toward D. galeata in sexual females and the opposite tendency in male production. One potential explanation for this difference could be in varying energy demands of sexual eggs and male production (Fig. 1). Ephippial production consumes energy reserves accumulated over two preceding instars (Lynch et al. 1986), whereas the production of males seems to be energetically similar to asexual propagation, as male and parthenogenetic progeny can be produced in the same clutch (Olmstead and Leblanc 2002). Further, we found that sexual reproduction can be additionally biased at the clonal level, as a single genotype dominated the whole male group for two successive seasons (Fig. 2). This is in accordance with studies on D. pulex, where clonal differences in contribution to sexual reproducetion have been

- 65 - Reproductive isolation Chapter 4 found (e.g., Innes and Dunbrack 1993) and suggest a general phenomenon in Daphnia. The next stage in successful hybridization is ‘mating’, which requires temporal and spatial co-occurrence of males and sexual females. In the D. galeata-hyalina-cucullata species complex hybridization is a regularly oc- curring process (e.g., Jankowski 2002), and taxon-specific diel vertical migration of sexual stages has not been detected within this complex (e.g., Spaak et al. 2004). Similarly, we also found in our Greifensee study, that most sexual females and males stay day and night in the uppermost 10 m of the water column, providing sufficient gender overlapping to enable mating (Fig. 3). 28. April 2005 2. May 2005 0 day nightday night 5

15 depth (m) 20

males 25 sexual females 30 0 0 0 0 50 50 50 50 100 100 100 100 proportion (%) Figure 3. Proportional depth distributions of males (open circles) and sexual females (filled circles) at midday (white background) and midnight (grey background) in Greifensee.

We detected many empty ephippia in the water column at the ‘egg development’-stage, which were most likely released by sexual females when the eggs were not fertilised (see also Keller and Spaak 2004). Empty ephippia have also been found in experimental crosses between D. galeata and D. cucullata (Schwenk et al. 2001), and in intraspecific crosses (e.g., D. magna, Boersma et al. 2000). It seems that empty ephippia are a common phenomenon in sexual reproduction in Daphnia. We found a significant relation between the maternal taxon and the proportion of empty ephippia. Hybrid genotypes had a larger proportion of empty ephippia, and thus a reduced sexual fitness, compared with the parental taxon D. galeata. The further reduction of hybrid fitness was confirmed by our finding that there were fewer viable ephippial

- 66 - Chapter 4 Reproductive isolation eggs with a hybrid genotype than expected by random mating (Fig. 4). This bias might be caused by problems with fertilization or egg development. Moreover, since parental taxa were identified with only three nuclear markers, individuals defined here as pure parentals may in fact have had a partial hybrid origin, and hence, the expected hybrid fraction among ephippial eggs is likely to be even larger. spring 2004 spring 2005

20 36 NA 20 37 78 27NA 80 100 pure D. galeata 80 hybrids

40 proportion (%) 20

0 males males hatchlings hatchlings ephippial eggs ephippial eggs sexual females sexual females expected offspring expected offspring

Figure 4. Proportional hybrid-parental distribution of sexual females (mothers), males (potential fathers), expected offspring, ephippial eggs (observed offspring), and hatchlings in Greifensee. For the assumptions for the calculation of expected offspring see text. The top of each bar indicates the number of analyzed individuals, NA refers to calculated proportions (see text).

In general, reproductive isolation mechanisms among taxa should most strongly affect the initial hybridization stages since F1 hybrids are the most difficult to be produced (reviewed by Mallet 2005). We found very low proportions of F1 hybrid hatchlings in hybridizing Daphnia, compared with backcrossed hybrid and D. galeata hatchlings. This reduced sexual fitness of

F1 hybrids limits their dispersal ability including colonization of new habitats, survival probability during harsh environmental conditions and thus heritability of genes as well as long-term persistence, and finally might lead to strong underestimation of historical hybrid occurrences based on diapausing egg bank analyzes. Analogous reductions in hybrid sexual fitness have been

- 67 - Reproductive isolation Chapter 4 described for other hybrid systems (e.g., Cruz et al. 2001), although they are not a general rule (e.g., Willi and Van Buskirk 2005). Therefore, we suggest that the F1 hybrid dominance often observed in asexual Daphnia populations (e.g., Keller and Spaak 2004) is maintained by asexual reproduction, which masks sexual hybrid inferiority. Hence, the cyclic parthenogenetic reproduction strategy of Daphnia seems to promote F1 hybrid success. The ability to reproduce both asexually and sexually is also common among many plants (e.g., Ellstrand et al. 1996) and is particularly important in two very common groups of freshwater zooplankton: cladocerans and mono- gonont rotifers (e.g., Hebert 1987). The frequency of asexual reproduction is often increased in hybrids, and thought to maintain hybrid populations (e.g., Ellstrand et al. 1996), even with very limited hybrid sexual fitness or sterility. Therefore, hybrids might be able to outperform their parents in both parental and hybrid habitats (e.g., Schwenk and Spaak 1995; Emms and Arnold 1997). Further, it has been proposed that asexual propagation can increase mating success in sparse populations (Gerritsen 1980). High asexual reproduction and reduced sexual fitness, as found here and in hybridizing Iris species (Johnston et al. 2004), might compensate rare hybrid production and reduced hybrid sexual fitness (Emms and Arnold 1997). We found indeed remarkable proportions of backcrossed hybrid hatchlings suggesting that the low sexual fitness of F1 hybrids in hybridizing Daphnia can be compensated generally through a high number of mating events. Asexual reproduction might therefore indirectly increase the probability that new unique hybrid genotypes will be created, due to an increased number of mating events. Under specific conditions, some of these new genotypes are obviously fitter than either parental type, leading to the widespread F1 hybrid dominance in hybridizing Daphnia taxa.

Acknowledgements We thank Esther Keller, Colleen Durkin and Richard Illi for support in the field and the laboratory. Morten Skage, Adam Petrusek, and Klaus Schwenk helped us with the ITS-RFLP marker. Further we thank Nelson Hairston Jr., Katja Räsänen and Christian Rellstab for useful comments on an earlier version of the manuscript. We thank Nelson Hairston Jr. for linguistic help.

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References Anderson, E., and G. L. Stebbins. 1954. Hybridization as an evolutionary stimulus. Evolution 8: 378-388. Anderson, E. C., and E. A. Thompson. 2002. A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160: 1217-1229. Arnold, M. L. 1992. Natural hybridization as an evolutionary process. Annu. Rev. Ecol. Syst. 23: 237-261. Arnold, M. L. 1997. Natural hybridization and evolution, 1st ed. Oxford University Press. Avise, J. C. 1994. Molecular markers, natural history and evolution, 1st ed. Chapman & Hall. Babik, W., J. M. Szymura, and J. Rafinski. 2003. Nuclear markers, mitochondrial DNA and male secondary sexual traits variation in a newt hybrid zone (Triturus vulgaris x T- montandoni). Mol. Ecol. 12: 1913-1930. Billiones, R., M. Brehm, J. Klee, and K. Schwenk. 2004. Genetic identification of Hyalodaphnia species and interspecific hybrids. Hydrobiologia 526: 43-53. Boersma, M., H. Boriss, and S. E. Mitchell. 2000. Maternal effects after sexual reproduction in Daphnia magna. J. Plankton Res. 22: 279-285. Brewer, M. C. 1998. Mating behaviours of Daphnia pulicaria, a cyclic parthenogen: comparisons with copepods. Philos. Trans. R. Soc. Lond. Ser. B-Biol. Sci. 353: 805- 815. Burke, J. M., M. R. Bulger, R. A. Wesselingh, and M. L. Arnold. 2000. Frequency and spatial patterning of clonal reproduction in Louisiana iris hybrid populations. Evolution 54: 137-144. Burke, J. M., S. E. Carney, and M. L. Arnold. 1998. Hybrid fitness in the Louisiana irises: Analysis of parental and F-1 performance. Evolution 52: 37-43. Butlin, R. 1987. Speciation by Reinforcement. Trends Ecol. Evol. 2: 8-13. Campbell, D. R., and N. M. Waser. 2001. Genotype-by-environment interaction and the fitness of plant hybrids in the wild. Evolution 55: 669-676. Carvalho, G. R., and H. G. Wolf. 1989. Resting eggs of lake-Daphnia. I. Distribution, abundance and hatching of eggs collected from various depths in lake sediments. Freshwat. Biol. 22: 459-470. Cruz, R., E. Rolan-Alvarez, and C. Garcia. 2001. Sexual selection on phenotypic traits in a hybrid zone of Littorina saxatilis (Olivi). J. Evol. Biol. 14: 773-785. De Meester, L. 1990. Genetisch-ecologische aspekten van fototaktisch gedrag bij Daphnia magna Straus. Ph.D. thesis. Rijksuniversiteit Gent. Dowling, T. E., and C. L. Secor. 1997. The role of hybridization and introgression in the diversification of animals. Annu. Rev. Ecol. Syst. 28: 593-619.

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Ellstrand, N. C., R. Whitkus, and L. H. Rieseberg. 1996. Distribution of spontaneous plant hybrids. Proc. Natl. Acad. Sci. U. S. A. 93: 5090-5093. Emms, S. K., and M. L. Arnold. 1997. The effect of habitat on parental and hybrid fitness: transplant experiments with Louisiana irises. Evolution 51: 1112-1119. Gerritsen, J. 1980. Sex and parthenogenesis in sparse populations. Am. Nat. 115: 718-742. Gießler, S. 1997. Analysis of reticulate relationships within the Daphnia longispina species complex. Allozyme phenotype and morphology. J. Evol. Biol. 10: 87-105. Hebert, P. D. N. 1985. Interspecific hybridization between cyclic parthenogens. Evolution 39: 216-220. Hebert, P. D. N. 1987. Genotypic characteristics of cyclic parthenogens and their obligately asexual derivates, p. 175-195. In S. Stearns [ed.], The evolution of sex and its consequences. Birkhäuser Verlag. Innes, D. J., and R. L. Dunbrack. 1993. Sex allocation variation in Daphnia pulex. J. Evol. Biol. 6: 559-575. Jankowski, T. 2002. From diapause to sexual reproduction: Evolutionary ecology of the Daphnia hybrid complex from Lake Constance. Ph. D. thesis. Univ. of Konstanz. Johnston, J. A., L. A. Donovan, and M. L. Arnold. 2004. Novel phenotypes among early generation hybrids of two Louisiana iris species: flooding experiments. J. Ecol 92: 967-976. Keller, B., H. R. Bürgi, M. Sturm, and P. Spaak. 2002. Ephippia and Daphnia abundances under changing throphic conditions. Verh. Internat. Verein. Limnol. 28: 851-855. Keller, B., and P. Spaak. 2004. Nonrandom sexual reproduction and diapausing egg production in a Daphnia hybrid species complex. Limnol. Oceanogr. 49: 1393– 1400. Lynch, M., L. J. Weider, and W. Lampert. 1986. Measurement of the carbon balance in Daphnia. Limnol. Oceanogr. 31: 17-33. Mallet, J. 2005. Hybridization as an invasion of the genome. Trends Ecol. Evol. 20: 229- 237.

Milne, R. I., S. Terzioglu, and R. J. Abbott. 2003. A hybrid zone dominated by fertile F1s: maintenance of species barriers in Rhododendron. Mol. Ecol. 12: 2719-2729. Moore, W. S. 1977. An evaluation of narrow hybrid zones in vertebrates. Q. Rev. Biol. 52: 263-277. Olmstead, A. W., and G. A. Leblanc. 2002. Juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna. J. Exp. Zool. 293: 736-739. Petrusek, A., F. Bastiansen, and K. Schwenk. 2005. European Daphnia Species (EDS) - Taxonomic and genetic keys. [Build 2006-01-12 beta]. CD-ROM, distributed by the authors. Department of Ecology and Evolution, J.W. Goethe-University, Frankfurt am Main, Germany & Department of Ecology, Charles University, Prague, Czechia.

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Rhymer, J. M., and D. Simberloff. 1996. Extinction by hybridization and introgression. Annu. Rev. Ecol. Syst. 27: 83-109. Schwenk, K., M. Bijl, and S. B. J. Menken. 2001. Experimental interspecific hybridization in Daphnia. Hydrobiologia 442: 67-73. Schwenk, K., and P. Spaak. 1995. Evolutionary and ecological consequences of interspecific hybridization in cladocerans. Experientia 51: 465-481. Skage, M., A. Hobæk, Š. Ruthová, B. Keller, A. Petrusek, J. Seća, and P. Spaak. submitted. Intragenomic sequence variation necessitates a new genetic method to distinguish species and hybrids in the Daphnia longispina complex. submitted to Hydrobiologia. Sokal, R. R., and F. J. Rohlf. 1995. Biometry, 3rd ed. W.H. Freeman and Co. Spaak, P. 1995. Sexual reproduction in Daphnia: interspecific differences in a hybrid species complex. Oecologia 104: 501-507. Spaak, P., A. Denk, M. Boersma, and L. J. Weider. 2004. Spatial and temporal patterns of sexual reproduction in a hybrid Daphnia species complex. J. Plankton Res. 26: 625– 635. Spaak, P., and J. R. Hoekstra. 1995. Life history variation and the coexistence of a Daphnia hybrid with its parental species. Ecology 76: 553-564. Stich, H. B., and W. Lampert. 1981. Predator evasion as an explanation of diurnal vertical migration by zooplankton. Nature 293: 396-398. Weider, L. J. 1993. Niche breadth and life history variation in a hybrid Daphnia complex. Ecology 74: 935-943. Willi, Y., and J. Van Buskirk. 2005. Genomic compatibility occurs over a wide range of parental genetic similarity in an outcrossing plant. Proc. R. Soc. Lond. B 272: 1333- 1338. Wolf, H. G., and M. A. Mort. 1986. Interspecific hybridization underlies phenotypic variability in Daphnia populations. Oecologia 68: 507-511. Wolinska, J., B. Keller, K. Bittner, S. Lass, and P. Spaak. 2004. Do parasites lower Daphnia hybrid fitness? Limnol. Oceanogr. 49: 1401-1407.

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CHAPTER 5

Anthropogenic impact on hybrid success and taxon niche separation in Daphnia with J. Wolinska, M. Manca and P. Spaak submitted to Ecology

Abstract Many European lakes have experienced rapid pollution increase (eutrophication) followed by slow recovery during the last century. Such habitat disturbances are known to facilitate hybridization. Although in general hybrids seem to perform poorly compared with their parental species, under specific environmental conditions they can be equally or more fit than their parents, which in turn can lead to hybrid dominance. Interspecific hybridization with hybrid dominance (i.e. F1 hybrids) is a common phenomenon in the Daphnia galeata-hyalina-cucullata species complex. Life-histories of parental taxa and

F1 hybrids have been compared in laboratory studies, but little is known about (abiotic) habitat preferences of various Daphnia taxa, or which conditions promote co-occurrence of parents and hybrids in natural populations. We used a multivariate approach to study taxon composition and habitat characteristics (i.e., lake size descriptors, elevation, total phosphorus concentration) of 43 Swiss and North Italian Daphnia lake assemblages to disentangle which conditions explain differences in community composition. We found that the Alps comprise two distinct biogeographic regions, with more D. galeata south of the Alps and more D. hyalina and D. cucullata north of the Alps. D. hyalina preferred large oligotrophic lakes, whereas D. galeata occurred predominantly at lower elevations (presumably a proxy for warmer temperatures). Further, we analyzed if the eutrophication history in 28 lakes might have an effect on recent F1 hybrid success. Indeed, F1 hybrid dominance could be explained by the magnitude of the change in phosphorus concentration over the past years. Albeit the cyclic parthenogenetic life cycle of Daphnia may facilitate the

- 73 - Hybrid superiority Chapter 5 outcome of hybridization, asexual propagation allows specific genotypes to reach high abundances without the complication of sexual reproduction. We suggest that human-mediated habitat disturbance (eutrophication and oligotrophication) and dispersal across natural barriers have facilitated hybridization and altered the Daphnia taxonomic composition of lakes. At the same time, specific habitat conditions might provide a refuge from hybridization for native genotypes.

Introduction Natural hybridization, resulting in individuals that are the result of crosses between genetically distinct populations of the same or different species (Arnold 1997), is a common phenomenon in both aquatic and terrestrial habitats (reviewed by Dowling and Secor 1997). Fitness of hybridizing taxa is influenced by environment-independent endogenous and environment-specific exogenous selection (reviewed by Burke and Arnold 2001). It has been shown that hybrids can cover a wide fitness range from poor to superior (reviewed by Rieseberg 1995; Barton 2001). In general, hybrids seem to perform poorly compared with their parents (Rieseberg 1995; Arnold 1997; Barton 2001). However, under specific environmental conditions hybrids can be equally or more fit than either parent (Rieseberg et al. 1996; Burke et al. 1998; Barton 2001). Three models exist to explain the coexistence of hybrid and parental taxa. The tension zone model (Key 1968; Barton and Hewitt 1985) assumes a balance between dispersal into the hybrid zone and selection against hybrids. In contrast, the bounded hybrid superiority model (Moore 1977; Moore and Koenig 1986) assumes that in intermediate environments, hybrids are fitter than either parent. Other zones are known in which hybrids are characterized by a mosaic-like distribution within heterogeneous habitats of parental species (mosaic model, Harrison 1985; Howard 1986; reviewed by Arnold 1997). Each patch has an unique set of environmental conditions that increase the probability that natural selection can pick out fitter genotypes, even though they might be rarely produced (Barton 2001). If the fitness of hybrids depends on environmental conditions, as the last two models propose, anthropogenic disturbance or fragmentation of natural systems can result in a widening, a nar- rowing, or the movement of hybrid zones. The extent of hybridization and the success of hybrid taxa can be increased among species that propagate both sexually and asexually (e.g., plants

- 74 - Chapter 5 Hybrid superiority with ramet production, cyclical parthenogenetic animals) because hybrid taxa can reach high abundances without the complication of sexual reproduction (e.g., Emms and Arnold 1997). Cyclical parthenogens, for which interspecific hybridization is regulatory observed, can be found within the genus Daphnia (Crustacea, Cladocera). Daphnia are the major herbivores in many lakes and ponds and, consequently, they can be important in aquatic food webs (Lampert et al. 1986). In many natural Daphnia assemblages, hybrids coexist with their parental species (Wolf and Mort 1986; Taylor and Hebert 1992; Schwenk and

Spaak 1995) and F1 hybrids are often the dominant taxon (Taylor and Hebert 1992; Spaak and Hoekstra 1995; Hobaek et al. 2004). Spaak and Hoekstra (1995) proposed the temporal hybrid superiority model to explain hybrid success in Daphnia. It has been shown that hybrids often combine the life- history traits of both parents in a beneficial way (Schwenk and Spaak 1995; Spaak 1995; Spaak et al. 2000), which might strengthen their competitive ability. Daphnia taxa are especially sensitive to factors altered by the trophic state of lakes such as predation pressure (Spaak and Hoekstra 1995; Kerfoot and Weider 2004), and food quantity (e.g., Demott et al. 2001) and quality (Hairston et al. 1999). Disturbance commonly facilitates success and the spread of hybrids (reviewed by Vilà et al. 2000; Kerfoot et al. 2004). Many lakes have experienced strong changes in their trophic state, resulting from anthropogenic eutrophication and subsequent oligotrophication during the last century. Eu- trophic conditions are known to disturb entire lake ecosystems. Hybrid dominance was found in several lakes with high trophic status (e.g., Spaak and Hoekstra 1997; Hobaek et al. 2004), but not, for example, in the relatively low trophic condition of Lake Constance (Jankowski and Straile 2004). The purpose of this study is to examine the fate of hybridizing Daphnia taxa in lakes with various trophic histories (over-fertilization of lakes in the last century followed by lake restoration efforts) with special emphasis on taxa occurrences and conditions leading to hybrid dominance.

Material and Methods Study sites & field sampling Twenty-six lakes in Switzerland and 17 lakes in Northern Italy inhabited by species from the Daphnia galeata-hyalina-cucullata species complex were screened for their Daphnia taxonomic composition. All lakes are at low elevations except the four Engadin lakes (CHAM, MURE, SEGL, SILV; for lakes abbreviations see Table 1) that are situated in a high elevation inner alpine

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Swiss valley. The lakes were sampled for zooplankton in spring and autumn (Northern Italy: May 2004 and September 2004; Switzerland: May-June 2003, August-October 2003 and August-October 2004). Samples from spring and autumn were pooled because we were interested in the overall Daphnia taxonomic composition and it is known that taxonomic composition in hybridizing Daphnia populations may vary seasonally (Spaak 1996; Jankowski and Straile 2004). Zooplankton samples were collected using a 250-μm mesh net (126-μm mesh for the Italian lakes) at the deepest part of the lake over the entire water column. Samples were cooled with ice during transport to the laboratory and either analysed subsequently or frozen for later allozyme analysis.

11 10 26 36 41 3 43 39 40 4 42 31 24 1 32 38 14 F hybrids 1 23 28 F hybrids 22 ghc 16 6 Pcuc 35 5 33 Pgal 29 Phyl 'not classifiable' 17

F1 hybrids 18 15 8 Fghc hybrids 21 Pcuc 19 37 9 25 12 Pgal 34 30 Phyl 20 27 'not classifiable' 7 2 13

Figure 1. Taxonomic composition of Daphnia asexual females across 43 analyzed lakes in northern Italy and Switzerland (2003 and 2004). Pie charts represent proportional occurrence of three parental taxa (Pgal = D. galeata; Phyl = D. hyalina; Pcuc = D. cucullata), F1 hybrids, Fghc hybrids and ‘not classifiable’ individuals (for details see the text). For lake abbreviations see Table 1.

Hybrid class identification Daphnia from all samples were assayed at four polymorphic allozyme loci using cellulose acetate electrophoresis (PGI, PGM, AAT, AO, for details see Keller and Spaak 2004). AO and AAT are diagnostic markers for D. galeata, D. hyalina and D. cucullata (Wolf and Mort 1986; Gießler 1997). All four

- 76 - Chapter 5 Hybrid superiority allozyme loci were used to identify hybrid classes with NewHybrid software (Anderson and Thompson 2002). This method uses Bayesian statistical methods to calculate the probability that an individual belongs to various hybrid categories. Since NewHybrid can only assign individuals to two hybridizing taxa at once, each lake population was divided into taxa pairs based on the two diagnostic allozyme loci. Individuals incorporating alleles from all three parental taxa were excluded from the analysis with NewHybrid and classified as Fghc hybrids. The identification probability in NewHybrid was set at 95%. The remaining individuals (probability <95%) were pooled and labelled as ‘not-classifiable’; most probably of hybrid origin. Individuals of parental taxa could appear in two different taxa pairs and therefore were analyzed twice. If they were identified once as a parental taxon and the other as ‘not classifiable’ they were incorporated to the parental groups. In this way we could discriminate between all three parental taxa (D. galeata Pgal,

D. hyalina Phyl and D. cucullata Pcuc) and F1, Fghc hybrids and the ‘not classifiable’ mixed group.

Abiotic lake descriptors, trophic state and lake history Hydrographic lake data and information about total phosphorus concentrations (Ptot) were collected from various sources (see Table 1). In lakes with no published total phosphorus concentration, Ptot was determined from water samples collected by integrative sampling of the upper 20 m. Furthermore, we used elevation as a proxy for water temperature (see: Livingstone et al. 1999; Li- vingstone et al. 2005). We used total phosphorus concentration (Ptot; μg L-1), lake volume (VO; 106 m3), maximum depth (DM; m), elevation (EL; m asl), and surface area (SU; km2) to investigate whether Daphnia taxa prefer different habitats (hereafter referred as ‘simple dataset’). Further, we were interested if these hydrographic parameters in combination with trophic lake history might explain frequently found F1 hybrid dominance. Therefore, we included the magnitude of phosphorus change since the maximum concentration ('Ptot/'t) to the analysis. This analysis (hereafter referred as ‘trophic history dataset’; Table 1) was applied to a subset of 28 lakes.

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Table 1. List with lake abbreviations and corresponding numbers for Figure 1, and the values of environmental variables used for statistical tests. Data sources: NA: no data available; a) Federal Office for the Environment (FOEN; P. Liechti); b) http://www.ise.cnr.it/LIMNO/LIMNO.htm; c) BUWAL (2002) d) Liechti (1994); e) Osservatorio dei Laghi Lombardi (2005); f) Ohlendorf (1998); g) Elber (2001); h) Ludovisi (2004); i) Garibaldi (1997); j) Keller (2002); k) Keller (2003); l) Ribi (2001); m) Kiefer (1987); n) pers. com. Herr Steiner NOK (Nordostschweizerische Kraftwerke); o) field sampling June/July 2003 upper 20m ) 3 ) m t) 2 6 ǻ ) ) -1 -1 Altitude asl) m (AL; References Volume Volume 10 (VO; Pmax Pmax L (μg years since ( Pmax Abbreviations Corresponding no (Fig. 1) Lake name North of the Alps South of the Alps Simple dataset' Trophic history dataset' Ptot L (μg Maximum depthMaximum m) (DM; area Surface km (SU; AGER 1 Ägerisee X X X 10 20-23 29 353 83 7.3 724 a, d, k ALSE 2 Lago di Alserio X X X 26 280 18 7 8.1 1.2 260 e, h BALD 3 Baldeggersee X X X 52 500-517 28 173 66 5.2 463 a, c, d BIEL 4 Bielersee X X X 13 140-132 33 1240 74 39.8 429 a, d BRIE 5 Brienzesee X X X 3 17-25 22 5170 261 29.8 564 a, c, d CHAM 6 Lej da Champfèr X X 11 NA NA 9 33 0.5 1791 f, o COMA 7 Lago di Comabbio X X X 72 200 28 17 8 3.6 243 b, e, h COMO 8 Lago di Como X X X 35 74 24 22500 410 145.0 198 e, h ENDI 9 Lago di Endine X X X 17 35 19 12 9 2.1 334 e, h, i GREI 10 Greifensee X X X 63 535-497 30 148 32.3 8.5 435 a, d, j HALL 11 Hallwillersee X X X 45 270-260 26 280 47 10.0 449 a, d IDRO 12 Lago di Idro X X 24 NA NA 684 122 11.4 370 b, e, h ISEO 13 Lago di Iseo X X X 17 41 23 7600 251 61.0 186 e, h KLON 14 Klöntalersee X X 12 NA NA 56 47 3.3 847 n, o LUGA 15 Lago di Lugano X X X 47 176 23 5860 288 48.9 271 d, e, h LUNG 16 Lungenersee X X 10 NA NA 66 68 2.0 689 l, o MAGG 17 Lago Maggiore X X X 11 35 25 37500 370 213.0 194 a, c, e, h MERG 18 Lago di Mergozzo X X 1 NA NA 83 73 1.8 194 b, h MONA 19 Lago di Monate X X 5 NA NA 45 34 2.5 266 e, h MONT 20 Lago di Montorfano X X X 8 15 4 2 7 0.5 397 e, h MORO 21 Lago Moro X X 8 NA NA 4 42 0.2 389 b, e, h MURE 22 Lej da S. Murezzan X X 43 NA NA 20 44 0.8 1768 f MURT 23 Murtensee X X X 27 155-147 23 550 45.5 22.8 429 a, d NEUE 24 Neuenburgersee X X X 14 59 23 13980 152 217.9 429 a, c, d ORTA 25 Lago di Orta X X 4 NA NA 1300 143 18.2 290 b, h PFAF 26 Pfäffikersee X X X 22 379-319 32 59 35 3.3 537 a, d, g PUSI 27 Lago di Pusiano X X X 74 200 12 69 24 5.0 259 e, h SARN 28 Sarnersee X X X 5 21 25 244 52 7.6 469 a, d SEGL 29 Lej da Segel X X 11 NA NA 137 71 4.1 1797 f SEGR 30 Lago del Segrino X X 12 NA NA 1 8.6 0.4 374 b, h SEMP 31 Sempachersee X X X 34 165 19 639 87 14.4 504 a, c, d SIHL 32 Sihlsee X X 13 NA NA 97 23 10.9 889 l, o SILV 33 Lej da Silvaplauna X X 13 NA NA 127 77 2.7 1791 f SIRI 34 Lago di Sirio X X 24 NA NA 5 43.5 0.3 271 b, h THUN 35 Thunersee X X X 3 21-23 22 6470 217 48.4 558 a, d TURL 36 Türlersee X X X 15 157 28 6 22 0.5 643 g, l VARE 37 Lago di Varese X X X 82 400 28 160 26 14.8 238 e, h VWS 38 Vierwaldstättersee X X X 7 32 27 11907 214 113.6 434 a, c, d WAGI 39 Wägitalersee X X 19 NA NA 149 65 4.2 900 m, o WALE 40 Walensee X X X 2 26-30 27 3180 145 24.1 419 a, d ZH_O 41 Zürichsee, Obersee X X X 14 41 31 467 48 20.3 406 a, d ZH_U 42 Zürichsee, Untersee X X X 25 130 33 3300 136 68.2 406 a, c, d ZUGE 43 Zugersee X X X 114 210-205 20 3174 198 38.3 413 a, d, l

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Statistical analyses Environmental parameters were log-transformed for normalization. Principal Component Analysis (PCA), Discriminant Analysis (DA), as well as ANOVAs were performed using STATISTICA for Windows, release 6.0 (StatSoft, Inc.). Correspondence Analysis (CA) and Canonical Correspondence Analysis (CCA) as well as following-up statistical tests were performed using 4.5 CANOCO package (Ter Braak and Šmilauer 2002). a) Community and taxon niche description We used PCA to detect major gradients in the environmental data set and for data reduction of abiotic (environmental) lake descriptors (‘simple dataset’). In case of co-linearity only those variables with highest factor coordinates were used for subsequent analyses. The Daphnia taxonomic composition dataset is based on relative proportions of each taxon and accordingly are compositional data containing many zeros. We incorporated therefore a CA and a CCA, because CA and its derivates (e.g. CCA) considers only the proportional relationship between variables and all differences between any data formats are removed (for details see Jackson 1997). Differences in PCA and CA sample scores of lakes north and south of the Alps along the first and second axis were tested with a one-way ANOVA. Then, we compared numbers of lakes with presence/absence of Fghc hybrids (detection level t1%) between the two biogeographic regions with a R × C test of independence (G-test, Sokal and Rohlf 1995). Further, we ran a CCA to detect patterns in the Daphnia taxonomic composition and environmental variables (‘simple dataset’). Statistical significance of the entire CCA and of the first two canonical axes was determined by Monte Carlo tests (999 permutations, Lepš and Šmilauer 2003). Forward selection (Ter Braak and Šmilauer 2002) was used to identify environmental variables explaining a statistically significant amount of variation in the Daphnia taxonomic composition by the inertia from marginal and conditional effects. Marginal effects explain the variation in the species data singly, whereas conditional effects show the amount of extra variation each variable contributes when added to the model (Ter Braak and Šmilauer 2002). Environmental variables, if significant for both marginal and conditional effects, were further used to detect coenoclines and to explain variation among taxa: generalized linear models were used (GLM with Poisson distribution and a logarithmic link function) (after Lepš and Šmilauer 2003). Smooth term complexity was selected with a nested model comparison (F statistics with P = 0.05 as threshold). We distinguished between monotonic

- 79 - Hybrid superiority Chapter 5 decreasing, left asymmetric, unimodal symmetric, right asymmetric, monotonic increasing, and u-shape (bimodal) type of response curves. Monotonic increasing and monotonic decreasing response curves always had their occurrence optima outside the measured variable ranges. The asymmetric, unimodal and bimodal response types have their occurrence optima within the measured ranges.

b) F1 hybrid superiority To test if the trophic lake history influenced the recent F1 hybrid dominance, we conducted a DA based on the ‘trophic history dataset’. We used a stepwise, forward selection procedure that sequentially selects the variable that makes the most significant additional contribution to the discrimination among tested groups (Statistica). To see how the chosen variables discriminate between lake populations, we performed a subsequent CA.

Results a) Community and taxon niche description PCA produced two main axes that explained 77.8% of the variance (PCA1: 55.9%; PCA2: 21.9%) and showed for PCA1 an eigenvalue of 2.794 and for PCA2 1.097. The first axis was mainly explained by lake size descriptors with strong co-linearity (factor coordinates for lake volume (VO) = -0.988; maximum depth (DM) = -0.9274; surface area (SU) = -0.945). Therefore we used only VO (largest factor coordinates) for subsequent analyses. The second axis was explained by total phosphorus concentration (Ptot = 0.824) and elevation (EL = -0.630). For the PCA scores, no significant differences were detected between lakes north and south of the Alps (PCA1: F1, 41 = 1.267,

P = 0.267; PCA2: F1, 41 = 0.538, P = 0.467).

Table 2. Environmental variables determined by forward selection in CCA in order of explained variability in Daphnia taxonomic composition (in order of their contribution to the entire model). Environmental variable abbreviations: EL = elevation (m asl), VO = lake volume (106 m3), and Ptot = total phosphorus concentration (Pg L-1), (significance level: P < 0.05).

Marginal effects Conditional effects Environmental variable Eigenvalue (O) F P Eigenvalue (O) F P EL 0.20 5.195 0.002 0.20 5.16 0.002 Ptot 0.14 3.595 0.016 0.10 3.00 0.029 VO 0.12 3.106 0.003 0.18 5.47 0.002

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In most lakes, F1 hybrids co-occurred with one or two parental taxa (Figure 1). D. Cohabitation of all three parental species was found in a single lake (ISEO, Nr. 13). Further, we found that Fghc hybrids were more common in northern lakes (G1 = 12.122; P < 0.001). CA produced two main axes that explained 76.8% of the Daphnia taxa variation (CA1: 45.1%; CA2: 31.7%) and had high eigenvalues (CA1: 0.793; CA2: 0.558). Lakes north and south of the

Alps were separated along CA1 (F1, 41 = 29.505, P < 0.0001); more Pgal in lakes south of the Alps and more Phyl and Pcuc in lakes north of the Alps. No discrimination between the two regions was found along CA2 (F1, 41 = 0.945, P = 0.337). The CCA discriminated between lakes north and south of the Alps and explained a significant amount of the weighted variance in the Daphnia taxonomic composition (Figure 2). Within lakes north of the Alps, the Engadin and lowland lakes were separated along EL. The first two axis of the CCA constrained by environmental variables were significant (CCA1: P = 0.002; CCA2: P = 0.047), explained 27.4% of the variance in the Daphnia dataset, and showed for CCA1 an eigenvalue of 0.358 and for CCA2 0.123. The first CCA axis was slightly more associated with VO, and the second axis with Ptot. The forward selection indicated that all three environmental variables were significantly related to the Daphnia taxonomic composition, with significant marginal and conditional effects (Table 2). The GLM-model for species responses to VO, EL and Ptot revealed that D. galeata occurred more often in lakes with a lower EL (F = 13.20, P = 0.001; monotonic decreasing curve). Further, D. galeata showed a u-shape response to VO, although not significant and the two main occurrences outside the measured VO range (F = 3.6; P = 3.60). D. hyalina occurred more frequently under low Ptot (F = 7.07, P = 0.011; monotonic decreasing curve) and in larger VO (F = 6.68, P = 0.013; monotonic increasing curve). For D. cucullata, no model could be fitted. F1 hybrids were solely influenced by Ptot (F = 3.26, P = 0.049; left asymmetric curve), and was found most frequently at 31.36 μg L-1 (SE: 0.072 μg L-1), which reflected slightly eutrophic conditions.

b) F1 hybrid superiority

The overall significant DA model (F3,24 =3.7089, P < 0.025) revealed that trophic history (i.e. 'P/'t) contributed significantly to the separation of ‘F1 hybrid dominated’ and ‘non-dominated’ populations (Partial Ȝ = 0.690, F = 10.759, P = 0.003). VO and EL were also incorporated in the model but

- 81 - Hybrid superiority Chapter 5 were not significant (VO: Partial Ȝ = 0.907, F = 2.452, P = 0.131; EL: Partial Ȝ = 0.954, F = 1.163, P = 0.292). With the fitted model, 5 lakes were misclassified (Figure 3). One significant canonical root (Wilks' Ȝ = 0.683; P = 0.025) could be extracted.

Figure 2. CCA results of Daphnia

1.0 communities and simple environmental variables in lakes north of the Alps (dots: low elevation lakes; diamonds: Engadin lakes in high elevation) and lakes south of the Alps (squares). Arrows point in the di- rection of increasing values for the respect- tive variables; arrow angles relative to axis

CCA2 and other variables indicate correlation strengths. Arrows represent environmental parameters (EL = elevation; VO = Vol- ume, and Ptot = total phosphorus concentration) and solid triangles symbolize re- south of the Alps lative proportions of various Daphnia taxa north of the Alps: low elevation Engadin -1.0 (Pgal = D. galeata; Phyl = D. hyalina; -1.0 CCA1 1.0 Pcuc = D. cucullata).

Discussion Population structure is usually influenced by both present and historical processes. Our results confirm this idea: past eutrophication has a long-lasting effect on the present F1 hybrid success across hybridizing Daphnia populations (Figure 3). We could further discriminate between taxa separated by biogeographic regions of the Alps; D. galeata more frequently dominated in southern whereas D. hyalina or D. cucullata dominated in northern lakes (Figures 1, 2). Invasion windows (i.e., proper colonization and long-term persistence) and dispersal-vector changes have been described as two processes that enable invasions to occur (Carlton 1996). Hence, the main factor promoting the formation of hybrids is human mediated dispersal (Vilà et al. 2000). Geo- graphic barriers, such as the Alps that have traditionally been considered as natural barriers for dispersal, have become porous due to human activity during the last century. This has been supported, for example, by the radiation history of D. parvula (reviewed by Panov et al. 2004). To colonize new habitats, first of all, a species needs to be able to cross geographical barriers. Diapausing eggs of Daphnia encased in a protective structure called ephippium, for example, are capable of long-distance dispersal mediated by

- 82 - Chapter 5 Hybrid superiority water birds and therefore affect gene flow between geographically isolated populations (Figuerola et al. 2005). On the other hand, remarkable genetic differences between neighbouring habitats have been found, suggesting limited dispersal or low colonization abilities (reviewed by De Meester et al. 2002). In hybridizing Daphnia populations, the presence of hybrids can be explained in two ways. Either hybrid taxa are locally produced, which requires co- occurrence of both parental species during certain time periods, or hybrids were produced elsewhere and subsequently colonize new habitats. Both mechanisms rely, however, on dispersal of diapausing eggs, either of parents or hybrids. Dispersal of species from the Daphnia galeata-hyalina-cucullata species complex is coupled with sexual reproduction and it has been shown that hybrids have a reduced sexual fitness (Keller and Spaak 2004; Keller et al. submitted). In contrast, D. galeata is a highly aggressive invader (Taylor et al. 2005). This suggests that D. galeata could have invaded lakes that were formerly inhabited by other species and that hybrids were locally produced. This scenario is supported by Spaak (1997), who found evidence for multiple hybridization events in natural Daphnia populations, and further has been shown already in Greifensee (Sandrock 2005) and Lake Constance (Weider et al. 1997; Jankowski and Straile 2003). When an individual reaches a new habitat, its ability to colonize it depends on its competitive ability under the present environmental conditions. Parental species and hybrids can occupy different ecological niches. Hybrids frequently have intermediate ecological tolerances (Bolton and Anderson 1987), but also often reveal novel ecological preferences, transcending those of parents and therefore can be found in novel or extreme habitats (reviewed by Levin 2004). Little is known about (abiotic) habitat preferences of various taxa of the D. galeata-hyalina-cucullata species complex, or about which factors promote co-occurrence of parents and hybrids in natural populations. In our study we found niche separation of parental species along specific environmental parameters (Figure 2). For example, D. hyalina is more likely to be found in large lakes with low total phosphorus concentrations and D. galeata occurs more often in warmer habitats at lower elevation. Presence of suitable environmental conditions does not guarantee successful colonization, because resident populations are normally buffered against invaders due to local adaptation and large population sizes (De Meester et al. 2002; Nosil et al. 2005). However, if resident populations are disturbed this might lead to ill- adapted populations and therefore increase the chance for successful invasion (Levin 2004).

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We found F1 hybrid dominance in lakes with the largest magnitude of total phosphorus change (Figure 3). This finding is consistent with the idea that hybrids perform better in highly disturbed habitats (e.g., Levin 2004). The transition from oligotrophic to eutrophic lake status (and vice versa) caused strong disturbance of entire lake ecosystems; in particular on phytoplankton biomass, species richness, and community composition (e.g., Seip and Reynolds 1995). This finding is in agreement with the proposed Temporal hybrid superiority model (Spaak and Hoekstra 1995), which explains hybrid success in Daphnia as a temporal phenomenon. Hence, our results are in concordance with historical taxa reconstructions of Greifensee and Lake Constance using DNA from diapausing egg banks (Brede 2003; Sandrock 2005). We propose, however, that the time horizon with suitable conditions for hybrids act on a longer time scale than the seasonal one proposed by Spaak and Hoekstra (1995).

'F1 hybrid dominated' 'F1 non-dominated'

large magnitude of missclassified small magnitude of phosphorus decrease by the model phosphorus decrease

-3 -2 -1 0 1 2 3 canonical root

Figure 3. Distribution of ‘F1 hybrid dominated’ (t50% F1 hybrids per lake) and ‘F1 non- dominated’ populations along extracted first canonical roots. Discrimination based on 28 lakes with information about trophic lake history (see ‘trophic history dataset’ in Table 1).

The final success of hybrids in a specific habitat depends on complex genotype-by-environment interactions (Arnold et al. 2001) as well as on stochastic effects related to extinction and colonization (Babik et al. 2003). When altered by disturbance, specific habitat conditions can change in such a way that the competitive abilities of resident genotypes are reduced, which leads to niche shifts of existing populations. This can also result in increased invasibility (reviewed by Levin 2004). Disturbance, however, can additionally alter the nature of species and enable hybridization by breaking down phenological barriers (Lamont et al. 2003). The success of hybrids is often facilitated and maintained by asexual or vegetative growth (e.g., Moody and Les 2002) that may ensure hybrid persistence and allow fit genotypes to reach high abundances without the cost of sexual reproduction (Burke et al. 2000). The importance of the cyclical parthenogenetic reproductive strategy in

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Daphnia should not be ignored as a factor that may stabilize hybrid lineages. Therefore, (asexual) fitness of Daphnia hybrid clones may alter competition among taxa and hence, may accentuate the influence of environmental factors on Daphnia populations. Daphnia from the D. galeata-hyalina-cucullata species complex have been used in numerous studies as model and target organisms. Based on our study of 43 populations, we conclude that the recent taxonomic composition can be predominantly explained by the two-fold effect of disturbance and dispersal; both were anthropogenically altered in the last century. Our results show that present-day F1 hybrid dominance is mainly the result of the magnitude of phosphorus concentration change in the past.

Acknowledgements We thank Christian Rellstab for providing us with zooplankton samples from Brienzersee and Esther Keller and Andrea Ferrari for collecting zooplankton samples from all other lakes. Colleen Durkin, Piotr Madej and Christoph Tellenbach helped run allozyme electrophoresis. Further, we thank Christoph Tellenbach for useful comments on an earlier version of the manuscript. We thank Chris Robinson and Nelson Hairston Jr. for linguistic help.

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Brede, N. 2003. Rekonstruktion populationsgenetischer Prozesse einer Invasion - Molekulare Analyse von Daphnia-Dauereiern aus Sedimentablagerungen des Bodensees. Diplomarbeit. Johann Wolfgang Goethe-Universität. Burke, J. M., and M. L. Arnold. 2001. Genetics and the fitness of hybrids. Annu. Rev. Gen. 35: 31-52. Burke, J. M., M. R. Bulger, R. A. Wesselingh, and M. L. Arnold. 2000. Frequency and spatial patterning of clonal reproduction in Louisiana iris hybrid populations. Evolution 54: 137-144. Burke, J. M., S. E. Carney, and M. L. Arnold. 1998. Hybrid fitness in the Louisiana irises: Analysis of parental and F-1 performance. Evolution 52: 37-43. Buwal. 2002. Die genutzte Umwelt - Fischerei: Lieber natürliche als gedüngte Fische, p. 221-226. Umwelt Schweiz 2002. BUWAL, Bundesamt für Statistik. Carlton, J. T. 1996. Pattern, process, and prediction in marine invasion ecology. Biological Conservation 78: 97-106. De Meester, L., A. Gomez, B. Okamura, and K. Schwenk. 2002. The Monopolization Hypothesis and the dispersal-gene flow paradox in aquatic organisms. Acta Oecologica 23: 121-135. Demott, W. R., R. D. Gulati, and E. Van Donk. 2001. Daphnia food limitation in three hypereutrophic Dutch lakes: Evidence for exclusion of large-bodied species by interfering filaments of cyanobacteria. Limnol. Oceanogr. 46: 2054-2060. Dowling, T. E., and C. L. Secor. 1997. The role of hybridization and introgression in the diversification of animals. Annu. Rev. Ecol. Syst. 28: 593-619. Elber, Hürlimann, and Niederberger. 2001. Entwicklung des Gesamtphosphors im Türlersee anhand der im Sediment eingelagerten Kieselalgen, p. 38. Baudirektion Kanton Zürich, Amt für Abfall, Wasser, Energie und Luft. Emms, S. K., and M. L. Arnold. 1997. The effect of habitat on parental and hybrid fitness: transplant experiments with Louisiana irises. Evolution 51: 1112-1119. Figuerola, J., A. J. Green, and T. C. Michot. 2005. Invertebrate eggs can fly: evidence of waterfowl-mediated gene flow in aquatic invertebrates. Am. Nat. 165: 274-280. Garibaldi, L., M. C. Brizzio, A. Varallo, and R. Mosello. 1997. The improving trophic conditions of Lake Endine (Northern Italy). Mem. Ist. Ital. Idrobiol. 56: 23-36. Gießler, S. 1997. Analysis of reticulate relationships within the Daphnia longispina species complex. Allozyme phenotype and morphology. J. Evol. Biol. 10: 87-105. Hairston, N. G., W. Lampert, C. E. Cáceres, C. L. Holtmeier, L. J. Weider, U. Gaedke, J. M. Fischer, J. A. Fox, and D. M. Post. 1999. Lake ecosystems - Rapid evolution revealed by dormant eggs. Nature 401: 446. Harrison, R. G. 1985. Pattern and process in a narrow hybrid zone. Heredity 56: 337-349.

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Hobaek, A., M. Skage, and K. Schwenk. 2004. Daphnia galeata x D. longispina hybrids in western Norway. Hydrobiologia 526: 55-62. Howard, D. J. 1986. A zone of overlap and hybridization between two ground cricket species. Evolution 40: 34-43. Jackson, D. A. 1997. Compositional data in community ecology: The paradigm or peril of proportions? Ecology 78: 929-940. Jankowski, T., and D. Straile. 2003. A comparison of egg-bank and long-term plankton dynamics of two Daphnia species, D. hyalina and D. galeata: Potentials and limits of reconstruction. Limnol. Oceanogr. 48: 1948-1955. Jankowski, T., and D. Straile. 2004. Allochronic differentiation among Daphnia species, hybrids and backcrosses: the importance of sexual reproduction for population dynamics and genetic architecture. J. Evol. Biol. 17: 312-321. Keller, B., H. R. Bürgi, M. Sturm, and P. Spaak. 2002. Ephippia and Daphnia abundances under changing throphic conditions. Verh. Internat. Verein. Limnol. 28: 851-855. Keller, B., and P. Spaak. 2004. Nonrandom sexual reproduction and diapausing egg production in a Daphnia hybrid species complex. Limnol. Oceanogr. 49: 1393– 1400. Keller, B., C. Tellenbach, J. Wolinska, and P. Spaak. submitted. Pre- and postzygotic reproductive isolation keep hybridizing Daphnia species distinct. Limnol. Oceanogr. Keller, P. 2003. Die Wasserqualität in den Zuger Gewässern von 1997 bis 2000. Umwelt Blickpunkt 20: 4-17. Kerfoot, W. C., X. Ma, C. S. Lorence, and L. J. Weider. 2004. Toward resurrection ecology: Daphnia mendotae and D. retrocurva in the coastal region of Lake Superior, among the first successful outside invaders? Journal Of Great Lakes Research 30: 285-299. Kerfoot, W. C., and L. J. Weider. 2004. Experimental paleoecology (resurrection ecology): Chasing Van Valen's Red Queen hypothesis. Limnol. Oceanogr. 49: 1300-1316. Key, K. H. L. 1968. Concept of stasipatric speciation. Systematic Zoology 17: 14-&. Kiefer, B. 1987. Untersuchungen zum Einfluss des Wasserregimes eines voralpinen Pumpspeicher-Sees (Wägitaler See) auf die Nährstoffversorgung der Phytoplanktonpopulation. Ph. D. thesis. Universität Zürich. Lamont, B. B., T. He, N. J. Enright, S. L. Krauss, and B. P. Miller. 2003. Anthropogenic disturbance promotes hybridization between Banksia species by altering their biology. J. Evol. Biol. 16: 551-557. Lampert, W., W. Fleckner, H. Rai, and B. E. Taylor. 1986. Phytoplancton control by grazing zooplankton: a study on the clear water phase. Limnol. Oceanogr. 31: 478- 490. Lepš, J., and P. Šmilauer. 2003. Multivariate Analysis of Ecological Data using CANOCO. Cambridge University Press.

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Levin, D. A. 2004. The ecological transition in speciation. New Phytol. 161: 91-96. Liechti, P. 1994. Der Zustand der Seen in der Schweiz. Schriftenreihe Umwelt 237: 162. Livingstone, D. M., A. F. Lotter, and H. Kettle. 2005. Altitude-dependent differences in the primary physical response of mountain lakes to climatic forcing. Limnol. Oceanogr. 50: 1313-1325. Livingstone, D. M., A. F. Lotter, and I. R. Walker. 1999. The decrease in summer surface water temperature with altitude in Swiss Alpine lakes: a comparison with air temperature lapse rates. Arct. Antarct. Alp. Res. 31: 341-352. Ludovisi, A., P. Pandolfi, and M. I. Taticchi. 2004. A proposed framework for the identification of habitat utilisation patterns of macrophytes in River Po catchment basin lakes (Italy). Hydrobiologia 523: 87-101. Moody, M. L., and D. H. Les. 2002. Evidence of hybridity in invasive watermilfoil (Myriophyllum) populations. Proc. Natl. Acad. Sci. U. S. A. 99: 14867-14871. Moore, W. S. 1977. An evaluation of narrow hybrid zones in vertebrates. Q. Rev. Biol. 52: 263-277. Moore, W. S., and W. D. Koenig. 1986. Comparative reproductive success of yellow- shafted, red-shafted and hybrid flickers across a hybrid zone. Auk 103: 42-51. Nosil, P., T. H. Vines, and D. J. Funk. 2005. Perspective: Reproductive isolation caused by natural selection against immigrants from divergent habitats. Evolution 59: 705-719. Ohlendorf, C. 1998. High Alpine lake sediments as chronicles for regional glacier and climate history in the Upper Engadine, southeastern Switzerland. Ph. D. thesis. ETH-Zürich. Osservatorio Dei Laghi Lombardi. 2005. Qualità delle acque lacustri in Lombardia - 1° Rapporto OLL 2004. Fondazione Lombardia per l’Ambiente e IRSA/CNR. Panov, V. E., P. I. Krylov, and N. Riccardi. 2004. Role of diapause in dispersal and invasion success by aquatic invertebrates. Journal of Limnology 63: 56-69. Ribi, B., H. Bührer, and H. Ambühl. 2001. Hypsographische Daten von Seen. Eawag. Rieseberg, L. H. 1995. The role of hybridization in evolution - old wine in new skins. Am. J. Bot. 82: 944-953. Rieseberg, L. H., B. Sinervo, C. R. Linder, M. C. Ungerer, and D. M. Arias. 1996. Role of gene interactions in hybrid speciation: Evidence from ancient and experimental hybrids. Science 272: 741-745. Sandrock, C. 2005. Rekonstruktion populationsgenetischer Prozesse bei Daphnia während des letzten Jahrhunderts im Greifensee - Introgressive Hybridisierung in Abhängigkeit von antropogenen verursachten Umweltveränderungen. Johann Wolfgang Goethe-Universität. Schwenk, K., and P. Spaak. 1995. Evolutionary and ecological consequences of interspecific hybridization in cladocerans. Experientia 51: 465-481.

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Seip, K. L., and C. S. Reynolds. 1995. Phytoplankton functional attributes along trophic gradient and season. Limnol. Oceanogr. 40: 589-597. Sokal, R. R., and F. J. Rohlf. 1995. Biometry, 3rd ed. W.H. Freeman and Co. Spaak, P. 1995. Cyclomorphosis as a factor explaining success of a Daphnia hybrid in Tjeukemeer. Hydrobiologia 307: 283-289. Spaak, P. 1996. Temporal changes in the genetic structure of the Daphnia species complex in Tjeukemeer, with evidence for backcrossing. Heredity 76: 539-548. Spaak, P. 1997. Hybridization in the Daphnia galeata complex: are hybrids locally produced? Hydrobiologia 360: 127-133. Spaak, P., and J. R. Hoekstra. 1995. Life history variation and the coexistence of a Daphnia hybrid with its parental species. Ecology 76: 553-564. Spaak, P., and J. R. Hoekstra. 1997. Fish predation on a Daphnia hybrid species complex: A factor explaining species coexistence? Limnol. Oceanogr. 42: 753-762. Spaak, P., J. Vanoverbeke, and M. Boersma. 2000. Predator induced life history changes and the coexistence of five taxa in a Daphnia species complex. Oikos 89: 164-174. Statistica. Electronic statistics textbook. StatSoft (http://www.statsoft.com/textbook/stathome.html). Taylor, D. J., and P. D. N. Hebert. 1992. Daphnia galeata mendotae as a cryptic species complex with interspecific hybrids. Limnol. Oceanogr. 37: 658-665. Taylor, D. J., H. L. Sprenger, and S. Ishida. 2005. Geographic and phylogenetic evidence for dispersed nuclear introgression in a daphniid with sexual propagules. Mol. Ecol. 14: 525–537. Ter Braak, C. J. F., and P. Šmilauer. 2002. CANOCO Reference manual and CanoDraw for Windows User’s guide: Software for Canonical Community Ordination (version 4.5). Microcomputer Power. Vilà, M., E. Weber, and C. M. D. Antonio. 2000. Conservation implications of invasion by plant hybridization. Biol. Invasions 2: 207-217. Weider, L. J., W. Lampert, M. Wessels, J. K. Colbourne, and P. Limburg. 1997. Long-term genetic shifts in a microcrustacean egg bank associated with anthropogenic changes in the Lake Constance ecosystem. Proc. R. Soc. Lond. B 264: 1613-1618. Wolf, H. G., and M. A. Mort. 1986. Interspecific hybridization underlies phenotypic variability in Daphnia populations. Oecologia 68: 507-511.

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CHAPTER 6

Concluding remarks and recommendations for further research

Discussion Lake sediments, as archives of historic population dynamics (Hairston et al. 1999b; Branvall et al. 2001; Hairston et al. 2001; Lotter et al. 2002; Hofmann 2003; Mergeay et al. 2004), help understand anthropogenic or naturally driven evolutionary and ecological processes over different time scales (Alekseev and Starobogatov 1996; Hairston and Kearns 2002). Based on their origin, there are two types of (sub)fossil records that can be found in and analyzed from lake sediments: preserved parts of animals / plants (e.g., bones, fossilized plant remains, carapax fragments, abdominal claws, diatom shells) and remains associated with asexual or sexual reproduction meant for dispersal (seeds, pollen, diapausing eggs). Seeds and ephippia (containing diapausing eggs of cladocerans) are often produced in large quantities and are well protected. As a consequence, they can stay viable in the sediment over long time periods (Hairston et al. 1999b; De Winton et al. 2000; Kerfoot and Weider 2004). The amount of sexually produced remains is not always correlated with density of organisms because their production is influenced by environmental conditions (e.g., Sarmaja-Korjonen 2004). Among plants, it was shown that some species are specialized in seed bank formation, whereas others are not (Bakker and Berendse 1999; Strykstra et al. 2002). For Daphnia egg banks, several studies showed a quantitative relationship between ephippia abundance and standing stock (Prazakova and Fott 1994; Verschuren and Marnell 1997). In Chapter 2, we also found a significant correlation between ephippia abundances in the egg bank and total phosphorus concentration used as proxy for past trophic lake conditions (eutrophication-oligotrophication) in the stratified sediment of Greifensee. However, with regular monthly monitoring of the pelagic popu-

- 91 - Discussion & Outlook Chapter 6 lation (i.e., quantifying sexual females) since the 1970s, no correlation was found between the abundance of sexual females and total phosphorus concentration. Environmental cues (i.e., seasonal and biotic) that induce diapause vary among (reviewed by Gyllstrom and Hansson 2004) and within zooplankton species (Tessier and Cáceres 2004). For instance, D. hyalina did not hatch from sediment layers of Lake Constance, although this taxon was present in the past (Weider et al. 1997; Jankowski and Straile 2003). The applicability of sexually produced ephippia in the D. galeata-hyalina-cucullata species complex for the reconstruction of former taxonomic composition was therefore questioned (Jankowski and Straile 2003). Nonetheless, species identification in the D. galeata-hyalina-cucullata complex is not possible based on carapax remains. Historical reconstructions therefore rely on hatched ex-ephippial daphniids or on DNA preserved in ephippial eggs: several PCR-based taxon affiliation methods exist for this species complex (see Introduction). We found in a hybridizing Daphnia population (Chapter 3) a strong bias in the taxonomic composition between the pelagic population and ex-ephippial hatchlings (from freshly-produced ephippial eggs as well as from older ones). To explain this mismatch, we studied reproductive isolation mechanisms between the species involved, and found incompatibilities with a reduced sexual fitness of D. galeata × hyalina hybrids (Chapter 4). Specifically, in the Greifensee, hybrids were rarely found among ex-ephippial hatchlings and ephippial eggs, although hybrids dominate the pelagic population. In some Daphnia populations, hybrids are extremely abundant and sometimes almost out-compete one or both parental species (e.g., Taylor and Hebert 1992; Spaak et al. 2004). Occurrences of hybrid dominances have been explained by the Temporary hybrid superiority model (Spaak and Hoekstra 1997). It also is known that parental species and hybrids of the D. galeata- hyalina-cucullata species complex do not form interpopulation transition zones (hybrid zones). Instead, rather patchy distributions of hybrids and parental species have been found across Europe (Schwenk and Spaak 1997). However, we found (Chapter 5) a biogeographic pattern with more D. galeata south of the Alps and D. hyalina / D. cucullata north of the Alps. Hence, hybrids were present on both sides of the Alps. Furthermore, it has been shown that taxa of the D. galeata-hyalina-cucullata species complex differ in their life history response to fish predation (e.g., Spaak et al. 2000) and food availability (Boersma and Vijverberg 1994). Both conditions (predation and food) are influenced by lake productivity (trophic lake state). These findings were the

- 92 - Chapter 6 Discussion & Outlook motivation for me to investigate if taxonomic composition and, specifically, hybrid dominance in natural Daphnia populations can be explained by trophic lake state and other lake descriptors. In Chapter 5, we found that lake volume, elevation (used as proxy for water temperature, see: Livingstone et al. 1999; Livingstone et al. 2005), and present total phosphorus concentration were sufficient to describe niche breadths between Daphnia taxa, but did not explain recent hybrid dominance. However, when incorporating the magnitude of phosphorus change as a proxy for habitat disturbance, recent hybrid dominance was explainable. Our results in Chapter 5 indicate that anthropogenically- caused lake pollution can influence present taxonomic composition in hybridizing Daphnia populations. In conclusion, we found evidence for reproductive isolation mechanisms between D. galeata and D. hyalina (Chapters 3-4). However, the presence of hybrids incorporating alleles from all three parental taxa (D. galeata, D. hyalina and D. cucullata) demonstrates the existence of intensive hybridization in this system (Chapter 5).

Outlook Hybridization is more common among plants than among animal species (reviewed by Mallet 2005). One explanation for this bias could be that plants have more possibilities to stabilize the breeding behaviour of hybrid lineages (reviewed by Rieseberg 1997). In Chapters 4 and 5, we speculated about how strongly asexual reproduction may influence the hybrid success in the cyclic parthenogen Daphnia. For that reason, we compared Daphnia with hybridizing plant species. Daphnia and many plant species share the capability for asexual reproduction as well as sexual reproduction combined with diapause and dormant seed production, respectively. Daphnia diapausing eggs encapsulated in ephippia can be seen as an equivalent to plant seeds (De Stasio 1989); both structures are produced sexually, meant for dispersal and dormancy, and hence are deposited in so-called seed or egg banks. In Daphnia, dispersal depends solely on ephippia. Whereas plants, in addition to seed dispersal, can exchange genetic material over long distances with pollen (Petit et al. 2004), facilitating hybridization. Enhanced (F1) hybrid asexual reproductive success, shown both for Daphnia (Chapter 4) and plant species (Ellstrand and Schierenbeck 2000;

Moody and Les 2002; Ayres et al. 2004), can cause F1 hybrid dominance and allow longevity, even when hybrids have a reduced sexual fitness. This might increase the probability for production of later hybrid generations (e.g., Fn hy-

- 93 - Discussion & Outlook Chapter 6 brids, backcrosses) (Burke et al. 2000). Indeed, we found in our lake survey (Chapter 5) a remarkable number of lakes with individuals incorporating alleles from all three parental species (i.e., Fghc hybrids). Fghc hybrids in natural Daphnia populations of the D. galeata-hyalina-cucullata species complex have been scarcely described; the only other records to our knowledge can be found in an egg bank analysis of Greifensee (Sandrock 2005).

The presence of Fghc hybrids suggests that many generations of hybridization events occurred within the D. galeata-hyalina-cucullata species complex: hybrid of one species pair had at least to mate with another species or with a hybrid of the third species. However, identification of hybrid genotypes relies strongly on the number of available molecular markers; an inadequate number of markers can veil hybrid occurrence and lead to misclassifications (Boecklen and Howard 1997). Consequently, taxon identification based on a small number of markers (as in our case four polymorphic allozyme markers and one ITS-RFLP marker) can underestimate the extent of hybridization and therefore related evolutionary processes. More precise taxon affiliations within the D. galeata-hyalina-cucullata species complex can be obtained with recently developed microsatellites (Brede et al. 2006). Molecular based markers in combination with a new method to induce Daphnia male production artificially (Rider et al. 2005) will allow the study of hybridization in Daphnia in more detail.

Extent of hybridization, reproductive isolation and hybrid fitness Reproductive isolation - In Chapter 3, we described a discrepancy in the taxonomic composition preserved in the egg bank and pelagic population of Greifensee, as has been previously found in Lake Constance (Weider et al. 1997; Brede 2003; Jankowski and Straile 2003). More precisely, after the invasion of D. galeata and subsequent hybridization with the native D. hyalina, D. hyalina could not be found in ephippial eggs. Notwithstanding, D. hyalina was present in the pelagic population. The question arises, therefore, if reproductive isolation mechanisms, as found in both lakes, depend on the extent of hybridization. To test how much the extent of hybridization affects the reproductive isolation between taxa, most recent parts of ephippial egg banks (used as a proxy for recently produced ephippia) can be compared with present pelagic populations of various hybridization extents and taxonomic compositions.

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F1 hybrid’s sexual fitness - We indirectly measured sexual fitness of D. galeata × hyalina hybrids (Chapter 4). We recorded the absence of D. galeata × hyalina hybrids in the recently produced ephippial eggs in a natural Daphnia population. It remains unresolved whether sexual fitness of D. galeata × hyalina hybrids is generally reduced. It has been shown for many species that hybridization is unidirectional, with one species serving as the maternal and the other one as the paternal species (reviewed by Wirtz 1999). I ran a preliminary analysis of maternal lines in the D. galeata-hyalina-cucullata species complex of 14 Swiss lakes (in total 223 D. galeata × hyalina hybrids were analyzed) comparing 16S rDNA (Schwenk et al. 1998) and found so far one undescribed haplotype. This haplotype most probably belongs to D. hyalina because it occurred exclusively in D. hyalina, D. galeata × hyalina and D. hyalina × cucullata hybrids (taxon affiliation based on AO / AAT and the classification method of Nason and Ellstrand (1993)). In addition, this genotype clustered with D. hyalina in an analysis of the mitochondrial COI fragment (Spaak and Keller, unpublished data). Although in hybridizing Daphnia, few populations with bidirectional mating were found, directionality was proposed (Schwenk 1993; Schwenk and Spaak 1995). We found bidirectionality in a preliminary analysis of mtDNA, but with a bias towards D. hyalina as the maternal species (~76% of all analyzed D. galeata × hyalina hybrids) (Keller and Spaak, unpublished data). In contradiction to other hybridizing species (reviewed by Wirtz 1999), Taylor and Hebert (1993) found that the maternal genome of hybridizing Daphnia belongs to the dominant parent, and suggested that this can be explained by environmentally-induced sexual reproduction. Our preliminary results for the Greifensee population contradict the finding of Taylor and Hebert (1993). D. hyalina, although hardly detectable in the recent pelagic population (Chapter 3), was the maternal taxon of 87% of all analyzed D. galeata × hyalina hybrids. An explanation for this finding might be that the recent situation in Greifensee is a relict of the historic situation when the native D. hyalina population was invaded by D. galeata as suggested by Sandrock (2005) or by D. galeata × hyalina hybrids with a D. hyalina mitochondrion. If hybridization occurred after the invasion among the more abundant maternal taxon (D. hyalina) and the rare paternal taxon (D. galeata), this would support the finding of Taylor and Hebert (1993). Later, the situation might have been conserved due to multiple hybridization events among predominantly D. galeata × hyalina hybrids (D. hyalina-mitochondrion) as maternal and D. galeata as paternal taxon. Moreover, the outcome of experimental crosses between D. galeata and D. cucullata revealed

- 95 - Discussion & Outlook Chapter 6 that crosses among D. galeata males and D. cucullata females were more successful than the reciprocal crosses (Schwenk et al. 2001). It might be that such biased fitness due to the maternal lineage influences the outcome of hybridization between D. galeata and D. hyalina as well; the observed reduced sexual fitness of F1 hybrids in Greifensee might be due simply to the fact that hybrids posses the mtDNA of D. hyalina and not of D. galeata. We hypothe- size that interspecific crosses (e.g., D. galeata × D. hyalina) result in lower numbers of viable ephippial eggs than intraspecific crosses (e.g., D. galeata ×

D. galeata) and that F2 hybrids are more difficult to obtain (i.e., lower number of viable eggs) than backcrosses to either parent. Moreover, we suppose that the fitness of crosses between D. galeata and D. hyalina depends on the maternal lineage, as shown for crosses between D. cucullata and D. galeata (Schwenk et al. 2001), but this needs further investigation. Crosses among specific Daphnia clones or species are difficult to achieve because a successful cross requires the simultaneous induction of both sexes. For this reason, direct comparisons of sexual fitness between parents and hybrids, as well as fitness of ex-ephippial hybrids with known kinship, were difficult to obtain and studies about this them are rare (e.g., Schwenk et al. 2001). Typical stimuli to induce sexual reproduction (male and sexually responsive females) are reduced food quality and quantity, decreasing photoperiod and increasing population density (e.g., Carvalho and Hughes 1983; Kleiven et al. 1992). Required stimuli can vary considerably for the induction of the two sexes as well as between taxa (Spaak 1995). A solution for controlled male induction might be the crustacean hormone methyl farnesoate, which is structurally similar to juvenoid (i.e. juvenile) hormones of insects and retinoid hormones of vertebrates. It has been successfully used to induce male development in several Daphnia species (Rider et al. 2005); four to six days after hormone application, 100% of all produced offspring were males (Dudycha, J. L. pers. com.).

Hybridization history and invasiveness History - Successful invasions of species that have colonized new regions within historical time followed by hybridization lead to ecological and evolutionary changes. We proposed that such an invasion happened within the D. galeata-hyalina-cucullata species complex in Greifensee (Chapter 5). Our argument is supported by paleogenetic surveys (Hairston et al. 1999a; Hairston et al. 2001) and ephippial analyses (Brede 2003; Jankowski and Straile 2003)

- 96 - Chapter 6 Discussion & Outlook of Daphnia from Lake Constance. Those studies showed that the lake was natively inhabited by D. hyalina, and later on invaded by D. galeata with subsequent hybridization. A comparable scenario was found for Greifensee based on egg bank analyses (Sandrock 2005). The question remains if the native species of lakes north of the Alps was always D. hyaline, whereas in lakes south of the Alps it was D. galeata (as we speculated in Chapter 5). To answer this question, the taxonomic composition of ephippial eggs obtained from sediment layers before eutrophication and hybridization started must be analysed and lakes north and south of the Alps must be compared with each other.

Invasiveness - It has been shown that D. galeata can be an aggressive invader (Taylor et al. 2005) and hybrids between D. galeata and D. hyalina can be quite successful (e.g., Chapter 5). If the ephippial egg bank data reveal that D. galeta was the invasive species in Swiss lakes, a subsequent question could be what caused these powerful invasions. Successful invasions occur regularly after multiple introductions and/or after a time lag (discussed in Facon et al. 2006), but the biological basis is not well understood. It has been proposed that invasiveness should be seen as an adaptive trait and could be promoted by (interspecific as well as intraspecific) hybridization and further catalyzed by human mediated dispersal and disturbance (Ellstrand and Schierenbeck 2000). Hence, three different scenarios were proposed, which singly or in combination might explain bioinvasions: migration behaviour changes, environmental changes, and evolutionary changes (Facon et al. 2006). Under the last scenario, bioinvasion starts as a result of genetic change (e.g., hybridization) in the invader. To determine the relative importance of each of the three scenarios, information about changes in the invaded environment, timing of invasion and migration regime is required. For hybridizing Daphnia populations, environmental changes (i.e. eutrophication-oligotrophication) of lakes are available (Chapter 5) or can be reconstructed from sediment cores. The timing of invasion can be reconstructed from ephippial egg banks (see earlier paragraph). Evolutionary divergence in adaptive traits between native (lakes south of the Alps) and introduced (lakes north of the Alps) populations may be tested with a quantitative genetic approach.

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Asexual reproduction, hybridization and taxonomic composition

F1 hybrid’s asexual fitness - We found, in a preliminary analysis, a significantly higher proportion of asexual gravid females among D. galeata × hyalina hybrids than among D. hyalina in two lakes with hybrid dominance (BIEL, HALL) in contrast to lakes with low hybrid occurrence (AGER, WAGI). But gravid females of the two taxa did not differ in their average clutch size (unpublished data Keller). However, an increased proportion of gravid F1 hybrid females, even when clutch sizes are comparable to the parental D. hyalina, can result in a larger birth rate. In a competition experiment between D. galeata and D. galeata × cucullata hybrids, it was shown that D. galeata quickly dominated the population due to its larger birth rate (Declerck and De Meester 2003). But the outcome of the competition changed when the daphniids were exposed to fish predators, which selectively preyed on the larger D. galeata. Thus, variable outcomes of taxa competition will lead to different temporal patterns in taxonomic composition. Two types of lakes inhabited by the D. hyalina-galeata-cucullata species complex can be distinguished: lakes with no clear seasonality in taxa occurrence like Greifen- see (Chapter 3), Plußsee (Spaak et al. 2004), or Tjeukemeer (Spaak 1996), and lakes with a distinct seasonal taxa succession, like Lake Constance (Jankowski and Straile 2004). It might be that in lakes with a-seasonal high occurrence of hybrids, which co-exist with none or only one parental taxon, that hybrids have occupied the ecological niche of the absent parental taxon. At what time scale do we need to consider “temporal” in the temporal hybrid superiority hypothesis: at the originally proposed seasonal time scale, or do we have to consider a much longer time scale as proposed in Chapter 5?

Questions about F1 hybrid fitness and performance can be tested in the field and experimentally. Life-history experiments could be applied to compare the performance of hybrids and parental taxa from ‘F1 hybrid dominated’ and ‘non-dominated’ lakes and a multivariate approach can be assessed to compare habitat characteristics between the two lake types. One possibility for ob- taining more precise information about environmental conditions might be to use total phosphorus concentration as a key factor; based on other biological variables expressing primary and secondary production (e.g., crustacean plankton, fish biomass) can be estimated or predicted (Håkanson and Boulion 2001). In addition, more precise physicochemical habitat characteristics of selected lakes should be collected (see Chapter VII in Schwenk 1997) to obtain a better understanding of habitat conditions.

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F1 hybrid’s over-wintering strategy - In cyclical parthenogens, the shift from asexual to sexual reproduction is environmentally induced and generally seen as a strategy to avoid unfavourable living conditions. Sexual reproduction increases diversity (genetic recombination) and is coupled with a two-fold dispersal tactic; dispersal in time (dormancy) and space (gene flow). Furthermore, it has been shown that asexual and sexual reproduction can simultaneously occur (Serra and King 1999). Environments, however, in which dormancy is not required might select against regular sex (Lynch 1984). In such a scenario prolonged asexual phases would then intensify clonal competition and selection. Clonal diversity would therefore decrease. In habitats populated by cyclical parthenogens (e.g., by aphids, rotifers and cladocerans) which do not require dormancy, there is no need for all clones to invest uniformly in sex (Green and Noakes 1995; Rispe and Pierre 1998). For example, the cereal aphid (Sitobion avenae) occurs in mixed populations of obligate and cyclical parthenogens, with decreasing sexuality southwards in climate permitting over wintering of nymphs and adults (Dedryver et al. 2001). Recently, an experiment with D. pulicaria clones sampled in habitats with different demands for dormancy/sexual reproduction revealed that populations differed significantly in their sex investment and 18% of all clones never invested in sexual reproduction, whereas 31% were gender specific in their sex allocation (Tessier and Cáceres 2004). Furthermore, the authors suggested a trade-off between the cost of diapause and the risk of remaining active. Indeed, Tessier and Cáceres (2004) found an ecological gradient in dormancy noticeable as a genetic gradient in sex induction and clonal variance. In Chapter 4 we also found a taxon specific tendency in the amount taxa invested in diapause and, further, that high abundance of genetically diverse F1 D. galeata × D. hyalina hybrids contrasts with their low sexual reproductive success. Diversity in Daphnia, however, is coupled with sex and can be explained in two ways; either by regular sexual reproduction or rare sexual reproduction in combination with long-time persistence of genetically diverse clonal lineages. Hence, it has been discussed that survival during unfavourable conditions is more likely if the numbers of individuals of a certain clone are high, which is more likely in large populations in eutrophic large lakes (De Meester et al. in press). Long-term survival in temporally variable environments, however, is only possible with a broad ecological tolerance; this might be the case for D. galeata × hyalina hybrids because they showed indis- tinguishable niche breadth from both parents (Weider 1993). However, our data suggest (Chapter 5) that in the D. galeata-hyalina-cucullata species com-

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plex parental species and F1 D. galeata × hyalina hybrids must have different over-wintering strategies and that F1 D. galeata × hyalina hybrids invest the least of all taxa into diapause/sexual reproduction. This hypothesis can be further tested in the field or experimentally, as shown for D. pulicaria populations (Cáceres and Tessier 2004; Tessier and Cáceres 2004). For the field survey, specific lakes inhabited by F1 D. galeata × D. hyalina hybrids and parental taxa (one or both) with different over wintering population densities could be compared.

Age of hybrid lineages - Based on the study of sexual fitness of D. galeata and D. galeata × hyalina hybrids in Greifensee (Chapter 4); we proposed that D. galeata × hyalina hybrids have a reduced sexual and diapause fitness. This finding contrasts with the pelagic hybrid dominance found in the six year population survey (Chapter 3). One explanation for such a scenario is that once established hybrids in the pelagic population are capable of long-term persistence under fluctuating abundances. Long-term persistence is inevitably accompanied by numerous mitotic cell divisions (i.e. amictic parthenogenesis) and therefore deleterious mutations accumulate. For cyclical parthenogens, simulations have shown that an increase in genetic load depends on the length of the asexual period (Palsson 2001). The question arises if hybrid lineages are on average older than parental lineages, and, speculatively, if a threshold for mutational load exists at which superiority might be reversed into inferiority and hence leads to a breakdown of these hybrid lineages. Under such a scenario, various lineages of F1 hybrids would co-occur with each other and might help to explain, for instance, the high variation in the susceptibility of hybrids to parasites (Wolinska 2006). It has been shown that obligately asexual lineages of D. pulex compared with sexual ones suffer a four times greater accumulation rate of deleterious amino acid substitutions in mitochondrial protein-coding genes (Paland and Lynch 2006). To obtain this result, Paland and Lynch (2006) compared rates of amino acid to silent substitutions, and amplified the entire mitochondrial genome of 15,333 bp length (see Crease 1999). Paland and Lynch (2006) summarized the advantages of mitochondrial protein-coding genes compared with nuclear genes for such kinds of analyses: (1) baseline mutation rate in animals is up to ten-fold higher in the mitochondrial genome, (2) animal mitochondrial proteins are in general mostly subject to purifying selection, (3) mildly deleterious amino-acid mutations arise in appreciable frequencies in

- 100 - Chapter 6 Discussion & Outlook mitochondria and participate significantly in evolutionary protein dynamics, (4) maternal inheritance ensures that the genes assayed have the same background mutation rate as silent sites, and (5) the haploid nature of mitochondria.

A target mitochondrial fragment to study mutational loads between F1 hybrids and parental daphniids might be ND5, a rapidly evolving gene (Colbourne et al. 1998) that has been used to compare the mutational load in asexual and sexual D. pulex lineages (Paland et al. 2005). Unfortunately primers working for Daphnia species other than D. pulex are not simply trans- ferable without difficulties to the D. galeata-hyalina-cucullata species complex (e.g., Brede et al. 2006, and Spaak and Keller unpublished data). Concluding remarks The research in this thesis has provided insight into the complex and extraordinary reproductive strategy, and the hybridization history of the genus Daphnia (i.e., D. galeata-hyalina-cucullata species complex) and has raised many questions that require future research. The increasing amounts of molecular markers (e.g., ITS-RFLP, microsatellites) will help to reduce the uncertainty in hybrid class affiliations. As a consequence, more complete insight into this hybridizing system will be provided and may help to quantify how strongly clonal reproduction is affecting the outcome of hybridization in Daphnia.

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Brede, N. 2003. Rekonstruktion populationsgenetischer Prozesse einer Invasion - Molekulare Analyse von Daphnia-Dauereiern aus Sedimentablagerungen des Bodensees. Diplomarbeit. Johann Wolfgang Goethe-Universität. Brede, N., A. Thielsch, C. Sandrock, P. Spaak, B. Keller, B. Streit, and K. Schwenk. 2006. Microsatellite markers for European Daphnia. Mol. Ecol. Notes 6: 536-539. Burke, J. M., M. R. Bulger, R. A. Wesselingh, and M. L. Arnold. 2000. Frequency and spatial patterning of clonal reproduction in Louisiana iris hybrid populations. Evolution 54: 137-144. Cáceres, C. E., and A. J. Tessier. 2004. Incidence of diapause varies among populations of Daphnia pulicaria. Oecologia 141: 425-431. Carvalho, G. R., and R. N. Hughes. 1983. The effect of food availability, female culture- density and photoperiod on ephippia production in Daphnia magna Straus (Crustacea: Cladocera). Freshwat. Biol. 13: 37-46. Colbourne, J. K., T. J. Crease, L. J. Weider, P. D. N. Hebert, F. Dufresne, and A. Hobæk. 1998. Phylogenetics and evolution of a circumarctic species complex (Cladocera : Daphnia pulex). Bio. J. Linn. Soc. 65: 347-365. Crease, T. J. 1999. The complete sequence of the mitochondrial genome of Daphnia pulex (Cladocera: Crustacea). Gene 233: 89-99. De Meester, L., J. Vanoverbeke, K. De Gelas, R. Ortells, and P. Spaak. in press. Genetic structure of cyclic parthenogenetic zooplankton populations – a conceptual framework. Arch. Hydrobiol. De Stasio, B. T., Jr. 1989. The seed bank of a freshwater crustacean: Copepodology for the plant ecologist. Ecology 70: 1377-1389. De Winton, M. D., J. S. Clayton, and P. D. Champion. 2000. Seedling emergence from seed banks of 15 New Zealand lakes with contrasting vegetation histories. Aquatic Botany 66: 181-194. Declerck, S., and L. De Meester. 2003. Impact of fish predation on coexisting Daphnia taxa: a partial test of the temporal hybrid superiority hypothesis. Hydrobiologia 500: 83- 94. Dedryver, C. A., M. Hullé, J. F. Le Gallic, M. C. Caillaud, and J. C. Simon. 2001. Coexistence in space and time of sexual and asexual populations of the cereal aphid Sitobion avenae. Oecologia 128: 379-388. Ellstrand, N. C., and K. A. Schierenbeck. 2000. Hybridization as a stimulus for the evolution of invasiveness in plants? Proc. Natl. Acad. Sci. U. S. A. 97: 7043-7050. Facon, B., B. J. Genton, J. Shykoff, P. Jarne, A. Estoup, and P. David. 2006. A general eco- evolutionary framework for understanding bioinvasions. Trends Ecol. Evol. 21: 130- 135.

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Green, R. F., and D. L. G. Noakes. 1995. Is a little bit of sex as good as alot. Journal of Theoretical Biology 174: 87-96. Gyllstrom, M., and L. A. Hansson. 2004. Dormancy in freshwater zooplankton: Induction, termination and the importance of benthic-pelagic coupling. Aquat. Sci. 66: 274- 295. Hairston, N. G., C. L. Holtmeier, W. Lampert, L. J. Weider, D. M. Post, J. M. Fischer, C. E. Cáceres, J. A. Fox, and U. Gaedke. 2001. Natural selection for grazer resistance to toxic cyanobacteria: Evolution of phenotypic plasticity? Evolution 55: 2203-2214. Hairston, N. G., and C. M. Kearns. 2002. Temporal dispersal: Ecological and evolutionary aspects of zooplankton egg banks and the role of sediment mixing. Integr. Comp. Biol. 42: 481-491. Hairston, N. G., W. Lampert, C. E. Cáceres, C. L. Holtmeier, L. J. Weider, U. Gaedke, J. M. Fischer, J. A. Fox, and D. M. Post. 1999a. Lake ecosystems - Rapid evolution revealed by dormant eggs. Nature 401: 446. Hairston, N. G., L. J. Perry, A. J. Bohonak, M. Q. Fellows, C. M. Kearns, and D. R. Engstrom. 1999b. Population biology of a failed invasion: Paleolimnology of Daphnia exilis in upstate New York. Limnol. Oceanogr. 44: 477-486. Håkanson, L., and V. V. Boulion. 2001. Regularities in primary production, secchi depth and fish yield and a new system to define trophic and humic state indices for lake ecosystems. International Review of Hydrobiology 86: 23-62. Hofmann, W. 2003. The long-term succession of high-altitude cladoceran assemblages: a 9000-year record from Sägistalsee (Swiss Alps). J. Paleolimn. 30: 291-296. Jankowski, T., and D. Straile. 2003. A comparison of egg-bank and long-term plankton dynamics of two Daphnia species, D. hyalina and D. galeata: Potentials and limits of reconstruction. Limnol. Oceanogr. 48: 1948-1955. Jankowski, T., and D. Straile. 2004. Allochronic differentiation among Daphnia species, hybrids and backcrosses: the importance of sexual reproduction for population dynamics and genetic architecture. J. Evol. Biol. 17: 312-321. Kerfoot, W. C., and L. J. Weider. 2004. Experimental paleoecology (resurrection ecology): Chasing Van Valen's Red Queen hypothesis. Limnol. Oceanogr. 49: 1300-1316. Kleiven, O. T., P. Larsson, and A. Hobæk. 1992. Sexual reproduction in Daphnia magna requires three stimuli. Oikos 65: 197-206. Livingstone, D. M., A. F. Lotter, and H. Kettle. 2005. Altitude-dependent differences in the primary physical response of mountain lakes to climatic forcing. Limnol. Oceanogr. 50: 1313-1325. Livingstone, D. M., A. F. Lotter, and I. R. Walker. 1999. The decrease in summer surface water temperature with altitude in Swiss Alpine lakes: a comparison with air temperature lapse rates. Arct. Antarct. Alp. Res. 31: 341-352.

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Lotter, A. F., P. G. Appleby, R. Bindler, J. A. Dearing, J. A. Grytnes, W. Hofmann, C. Kamenik, A. Lami, D. M. Livingstone, C. Ohlendorf, N. Rose, and M. Sturm. 2002. The sediment record of the past 200 years in a Swiss high- alpine lake: Hagelseewli (2339 ma.s.l.). J. Paleolimn. 28: 111-127. Lynch, M. 1984. The genetic structure of a cyclical parthenogen. Evolution 38: 186-203. Mallet, J. 2005. Hybridization as an invasion of the genome. Trends Ecol. Evol. 20: 229- 237. Mergeay, J., D. Verschuren, L. Van Kerckhoven, and L. De Meester. 2004. Two hundred years of a diverse Daphnia community in Lake Naivasha (Kenya): effects of natural and human-induced environmental changes. Freshwat. Biol. 49: 998-1013. Moody, M. L., and D. H. Les. 2002. Evidence of hybridity in invasive watermilfoil (Myriophyllum) populations. Proc. Natl. Acad. Sci. U. S. A. 99: 14867-14871. Nason, J. D., and N. C. Ellstrand. 1993. Estimating the frequencies of genetically distinct classes of individuals in hybridized populations. Journal of Heredity 84: 1-12. Paland, S., J. K. Colbourne, and M. Lynch. 2005. Evolutionary history of contagious asexuality in Daphnia pulex. Evolution 59: 800-813. Paland, S., and M. Lynch. 2006. Transitions to asexuality result in excess amino acid substitutions. Science 311: 990-992. Palsson, S. 2001. The effects of deleterious mutations in cyclically parthenogenetic organisms. Journal of Theoretical Biology 208: 201-214. Petit, R. J., C. Bodénès, A. Ducousso, G. Roussel, and A. Kremer. 2004. Hybridization as a mechanism of invasion in oaks. New Phytol. 161: 151-164. Prazakova, M., and J. Fott. 1994. Zooplankton decline in the Cerne Lake (Sumava Mountains, Bohemia) as reflected in the stratification of cladoceran remains in the sediment. Hydrobiologia 274: 121-126. Rider, C. V., T. A. Gorr, A. W. Olmstead, B. A. Wasilak, and G. A. Leblanc. 2005. Stress signaling: coregulation of hemoglobin and male sex determination through a terpenoid signaling pathway in a crustacean. J Exp Biol 208: 15-23. Rieseberg, L. H. 1997. Hybrid origins of plant species. Annu. Rev. Ecol. Syst. 28: 359-389. Rispe, C., and J. S. Pierre. 1998. Coexistence between cyclical parthenogens, obligate parthenogens, and intermediates in a fluctuating environment. Journal of Theoretical Biology 195: 97-110. Sandrock, C. 2005. Rekonstruktion populationsgenetischer Prozesse bei Daphnia während des letzten Jahrhunderts im Greifensee - Introgressive Hybridisierung in Abhängigkeit von antropogenen verursachten Umweltveränderungen. Johann Wolfgang Goethe-Universität. Sarmaja-Korjonen, K. 2004. Chydorid ephippia as indicators of past environmental changes - a new method. Hydrobiologia 526: 129-136.

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Schwenk, K. 1993. Interspecific hybridization in Daphnia: distinction and origin of hybrid matrilines. Mol. Biol. Evol. 10: 1289-1302. Schwenk, K. 1997. Evolutionary genetics of Daphnia species complexes - hybridism in syntopy. Ph.D. University of Utrecht. Schwenk, K., M. Bijl, and S. B. J. Menken. 2001. Experimental interspecific hybridization in Daphnia. Hydrobiologia 442: 67-73. Schwenk, K., A. Sand, M. Boersma, M. Brehm, E. Mader, D. Offerhaus, and P. Spaak. 1998. Genetic markers, genealogies and biogeographic patterns in the cladocera. Aquatic Ecology 32: 37-51. Schwenk, K., and P. Spaak. 1995. Evolutionary and ecological consequences of interspecific hybridization in cladocerans. Experientia 51: 465-481. Schwenk, K., and P. Spaak. 1997. Ecological and genetics of interspecific hybridization in Daphnia, p. 199-229. In B. Streit, T. Städler and C. M. Lively [eds.], Evolutionary ecology of freshwater animals. Birkhäuser Verlag. Serra, M., and C. E. King. 1999. Optimal rates of bisexual reproduction in cyclical parthenogens with density-dependent growth. J. Evol. Biol. 12: 263-271. Spaak, P. 1995. Sexual reproduction in Daphnia: interspecific differences in a hybrid species complex. Oecologia 104: 501-507. Spaak, P. 1996. Temporal changes in the genetic structure of the Daphnia species complex in Tjeukemeer, with evidence for backcrossing. Heredity 76: 539-548. Spaak, P., A. Denk, M. Boersma, and L. J. Weider. 2004. Spatial and temporal patterns of sexual reproduction in a hybrid Daphnia species complex. J. Plankton Res. 26: 625– 635. Spaak, P., and J. R. Hoekstra. 1997. Fish predation on a Daphnia hybrid species complex: A factor explaining species coexistence? Limnol. Oceanogr. 42: 753-762. Spaak, P., J. Vanoverbeke, and M. Boersma. 2000. Predator induced life history changes and the coexistence of five taxa in a Daphnia species complex. Oikos 89: 164-174. Strykstra, R. J., R. M. Bekker, and J. Van Andel. 2002. Dispersal and life span spectra in plant communities: a key to safe site dynamics, species coexistence and conservation. Ecography 25: 145-160. Taylor, D. J., and P. D. N. Hebert. 1992. Daphnia galeata mendotae as a cryptic species complex with interspecific hybrids. Limnol. Oceanogr. 37: 658-665. Taylor, D. J., and P. D. N. Hebert. 1993. Habitat-dependent hybrid parentage and differential introgression between neighboringly sympatric Daphnia species. Proc. Natl. Acad. Sci. U. S. A. 90: 7079-7083. Taylor, D. J., H. L. Sprenger, and S. Ishida. 2005. Geographic and phylogenetic evidence for dispersed nuclear introgression in a daphniid with sexual propagules. Mol. Ecol. 14: 525–537.

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Tessier, A. J., and C. E. Cáceres. 2004. Differentiation in sex investment by clones and populations of Daphnia. Ecol. Lett. 7: 695-703. Verschuren, D., and L. F. Marnell. 1997. Fossil zooplankton and the historical status of westslope cutthroat trout in a headwater lake of Glacier National Park, Montana. Transactions of the American Fisheries Society 126: 21-34. Weider, L. J. 1993. Niche breadth and life history variation in a hybrid Daphnia complex. Ecology 74: 935-943. Weider, L. J., W. Lampert, M. Wessels, J. K. Colbourne, and P. Limburg. 1997. Long-term genetic shifts in a microcrustacean egg bank associated with anthropogenic changes in the Lake Constance ecosystem. Proc. R. Soc. Lond. B 264: 1613-1618. Wirtz, P. 1999. Mother species-father species: unidirectional hybridization in animals with female choice. Animal Behaviour 58: 1-12. Wolinska, J. 2006. Parasites and predators structure hybridizing Daphnia communities. Ph.D. thesis. ETHZürich.

- 106 - CURRICULUM VITAE Name Barbara Keller Date of birth 16.01.1972 Nationality Swiss

Education 2002-2006, Ph. D. thesis at the Department of Aquatic Ecology, Eawag/ETH Zürich, Switzerland, under supervision of PD Dr. Piet Spaak, Zürich “Extent of hybridization and reproductive isolation in Daphnia”

1999, Diploma thesis at the Swiss Federal Institute of Technology Zürich, Switzerland (ETH Zürich/Eawag), under supervision of Prof. James V. Ward and Dr. Klement Tockner “Struktur und Zoobenthosdiversität von zwei Schwemmebenen im Oberlauf des Tagliamento-Flusses im Friaul (Italien)”

1995-2000, Study of ‘Systematische und ökologische Biologie’ at the Swiss Federal Institute of Technology Zürich (ETH Zürich)

Teaching 1999-present, Teaching assistance at the Department of experience Aquatic Ecology of the Swiss Federal Institute of Aquatic Science and Technology (Eawag/ETH Zürich, Switzerland)

Other Organization of the 7th International Symposium on Cladocera, activities Herzberg, Switzerland, 2005

Referee for Limnology and Oceanography, Annales de Limnology and Hydrobiologia

2003-2006, Introductory lectures, support and maintenance to/of the EndNote-library-database of the Swiss Federal Institute of Aquatic Science and Technology (Eawag)

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Conference as presenting author: presentations “Can Daphnia populations be reconstructed from diapausing eggs?” Keller, B., Wolinska, J. and P. Spaak; 7th International Symposium on Cladocera, Herzberg, Switzerland, 2005 (oral)

“Extent of hybridization in a Daphnia species complex” Keller, B., and P. Spaak; 10th Congress of the European Society for Evolutionary Biology (ESEB), Krakóv, PL, 2005 (oral)

“Do Daphnia hybrids invest in diapause?” Keller, B. and P. Spaak; 4th International Symposium of Ecological Genetics, Antwerp, Belgium, 2005 (oral)

“Daphnia hybrids do not invest in diapause” Keller, B. and P. Spaak; 29th Congress of the International Association of Theoretical and Applied Limnology (SIL), Lahti, Finland, 2004 (oral)

“Sexual reproduction in a Daphnia hybrid species complex“ Keller, B., Wolinska, J. and P. Spaak; Biology04, Annual Meeting of the Swiss Zoological, Botanical and Mycological Societies, Freiburg, Switzerland, 2004 (poster)

“Non-random sexual reproduction in a Daphnia hybrid species complex: its consequences for the diapausing egg bank” Keller, B. and P. Spaak; 9th Congress of the European Society for Evolutionary Biology, Leeds, UK, 2003 (poster)

“Do ephippia from lake sediment represent the past pelagic population?” Keller, B. and P. Spaak; 3rd International Symposium of Ecological Genetics, Leuven, Belgium, 2003 (oral)

“Do resting eggs represent the pelagic population?” Keller, B. and P. Spaak; Biology03, Annual Meeting of the Swiss Zoological, Botanical and Mycological Societies, Zuerich, Switzerland, 2003 (oral)

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“How well does a resting egg bank reflect its corresponding pelagial Daphnia population?” Keller, B. and P. Spaak; 8th Meeting of PhD Students in Evolutionary Biology, Lohja, Finland, 2002 (oral)

“How well does a resting egg bank reflect its corresponding pelagial Daphnia population?” Keller, B. and P. Spaak; 7th International Symposium on Cladocera, Wierzba, Poland, 2002 (oral)

“Ephippia and clonal/taxon distribution in space and time in a Daphnia species complex” Keller, B., Spaak, P., and H. R. Bürgi; 7th Meeting of PhD Students in Evolutionary Biology, Bernried, Germany, 2001 (oral)

“Which processes determine genetic differences between pelagic Daphnia and their egg bank?” Keller, B., Spaak, P., and H. R. Bürgi; 2nd International Symposium of Ecological Genetics, Antwerp, Belgium, 2001 (oral)

as co-author: “Hybridization of the habitat: Qualitative habitat change is associated with interspecific hybridization in Daphnia“ Brede, N., Sandrock, C., Keller, B., Spaak, P., Straile, D., and K. Schwenk; 7th International Symposium on Cladocera, Herz- berg, Switzerland, 2005 (oral)

“The evolutionary history of interspecific hybridization in Daphnia” Sandrock, C., Brede, N., Keller, B., Spaak, P., and K. Schwenk; 7th International Symposium on Cladocera, Herz- berg, Switzerland, 2005 (poster)

“New microsatellite markers for the European Daphnia longispina group: development and applications“ Thielsch, A., Brede, N. Sandrock, C., Petrusek, A., Keller, B., Spaak, P., Streit, B., and K. Schwenk; 7th International Sym- posium on Cladocera, Herzberg, Switzerland, 2005 (poster)

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“Hybridization - an instrument of evolutionary changes in species” Brede, N., C. Sandrock, D. Straile, B. Keller, P. Spaak and K. Schwenk; 4th International Symposium of Ecological Genetics, Antwerp, Belgium, 2005 (poster)

“Parasites lower Daphnia hybrids fitness” Wolinska, J., Keller, B., Bittner, K., Lass, S., and P. Spaak; 9th Congress of the European Society for Evolutionary Biology, Leeds, UK, 2003 (poster)

“Selective parasitism infection load of a Daphnia hybrid species complex” Wolinska, J., Keller, B., and P. Spaak; 3rd International Sym- posium of Ecological Genetics, Leuven, Belgium, 2003 (oral)

“Parasitism as a factor which lowers Daphnia hybrid fitness” Wolinska, J., Keller, B., and P. Spaak; Biology03, Annual Meeting of the Swiss Zoological, Botanical and Mycological Societies, Zuerich, Switzerland, 2003 (oral)

“Why are the asexual Daphnia population in the lake and their ‘resting’ sexual descendants in the sediment different?” Spaak, P., Keller, B., and M. Rudis; 8th Congress of the European Society for Evolutionary Biology, Aarhus, Denmark, 2001 (oral)

“Occurrence of Daphnia hybrid ephippia in the sediment compared to the lake” Spaak, P., Keller, B., and H. R. Bürgi; 28th congress of the Societas Internationalis Limnologiae, Melbourne, Australia, 2001 (oral)

“Egg bank biology – Daphnia hybrids are not as dominant in the sediment compared to the lake” Spaak, P., Keller, B., and H. R. Bürgi; Annual meeting of the American Society of Limnology and Oceanography, Copenhagen, Denmark, 2000 (poster)

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Publications Skage, M., A. Hobæk, Š. Ruthová, B. Keller, A. Petrusek, J. Seća, and P. Spaak. in revision. Intragenomic sequence variation necessitates a new genetic method to distinguish species and hybrids in the Daphnia longispina complex. In revision, Hydrobiologia.

Keller, B., J. Wolinska, C. Tellenbach, and P. Spaak. 2007. Reproductive isolation keeps hybridizing Daphnia species distinct. In press, Limnology and Oceanography.

Spaak, P., and B. Keller. 2006. Diapause and its consequences in the Daphnia galeata – cucullata – hyalina species complex. In press in V. Alekseev and B. De Stasio, editors. Diapause in aquatic invertebrates: role for ecology, physiology and human uses. Springer.

Wolinska, J., B. Keller, M. Manca, and P. Spaak. 2006. Parasite survey of a Daphnia hybrid complex: host-specificity and environment determine infection. Journal of Animal Ecology.

Brede, N., A. Thielsch, C. Sandrock, P. Spaak, B. Keller, B. Streit, and K. Schwenk. 2006. Microsatellite markers for European Daphnia. Molecular Ecology Notes 6:536-539.

Keller, B., J. Wolinska, and P. Spaak. 2005. Daphnia hybrids do not invest in diapause. Verhandlungen Internationale Vereinigung für Theoretische und Angewandte Limnologie 29:226.

Wolinska, J., B. Keller, and P. Spaak. 2005. Parasite - host preference in hybrid Daphnia populations. Verhandlungen Internationale Vereinigung für Theoretische und Angewandte Limnologie 29:340-341.

Keller, B., and P. Spaak. 2004. Nonrandom sexual reproduction and diapausing egg production in a Daphnia hybrid species complex. Limnology and Oceanography 49:1393–1400.

Wolinska, J., B. Keller, K. Bittner, S. Lass, and P. Spaak. 2004. Do parasites lower Daphnia hybrid fitness? Limnology and Oceanography 49:1401-1407.

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Spaak, P., and B. Keller. 2004. No evidence for adaptive micro-evolution to a decrease in phosphorus-loading of a Daphnia population inhabiting a pre-alpine lake. Hydrobiologia 526:15-21.

Löffler, A., J. Wolinska, B. Keller, K. O. Rothhaupt, and P. Spaak. 2004. Life history patterns of parental and hybrid Daphnia differ between lakes. Freshwater Biology 49: 1372– 1380.

Arscott, D. B., B. Keller, K. Tockner, and J. V. Ward. 2003. Habitat structure and Trichoptera diversity in two headwater flood plains, NE Italy. International Review of Hydrobiology 88:255–273.

Bürgi, H. R., H. Buehrer, and B. Keller. 2003. Long-term changes in functional properties and biodiversity of plankton in Lake Greifensee (Switzerland) in response to phosphorus. Aquatic Ecosystem Health and Management 6:147-158.

Keller, B., H. R. Bürgi, M. Sturm, and P. Spaak. 2002. Ephippia and Daphnia abundances under changing throphic conditions. Verhandlungen Internationale Vereinigung für Theoretische und Angewandte Limnologie 28:851-855.

- 112 - DANKSAGUNG

Ohne die tatkräftige Hilfe und umfangreiche Unterstützung einer ganzen Reihe von Leuten wäre diese Arbeit nicht möglich gewesen. Deshalb möchte ich an dieser Stelle allen im Folgenden genannten Personen ein grosses MERCI aussprechen.

Zuerst möchte ich meinem Betreuer Piet Spaak danken, der mich mit seinem Angebot, neben der Tätigkeit als Unterrichtsassistentin Forschung zu betreiben, in die Welt der Daphnien (Wasserflöhe) und Evolutionsbiologie eingeführt hat. Piet hat mir meine (Teilzeit-)Forschung ermöglicht, während der ich meine Tätigkeiten, meine Energie und meinen Enthusiasmus zwischen Forschung, Lehre und Bibliotheksarbeit auftteilen und zu einer (Gesamtheits-) Doktorarbeit zusammenführen konnte - herzlichsten Dank!

Prof. James V. Ward hat es mir ermöglicht, meine Arbeit in der Abteilung Limnologie an der EAWAG zu beginnen, und Prof. Jukka Jokela in der zwischenzeitlich zu Aquatic Ecology (Eawag) umbenannten Abteilung abzuschliessen.

Danke Hansruedi Bürgi, der während diversen ETH-Praktika auf dem Greifensee meinen Enthusiasmus für die Daphnien und deren Ephippien geteilt hat. So konnte ich viele Probenahmen mit StudentInnen-Praktika kombinieren.

Nelson Hairston danke ich für die Bereitschaft zur Begutachtung der Arbeit. Zudem hat er es mir zusammen mit Piet ermöglicht, an der Cornell University in Ithaca den Umgang mit einem Sequenzer zu erlernen.

Ein ganz speziell grosses MERCI an Esther Keller, Justyna Wolinska und Christoph Tellenbach, konnte ich doch zu allen (Un-)Zeiten ihre Hilfe und ihren Rat in Anspruch nehmen. Was sehr erstaunlich ist: Sie haben sich nie über meine möglichen und unmöglichen Ideen gewundert und mich immer bei deren Ausarbeitung und Umsetzung unterstützt!

Meinen herzlichen Dank verdienen Esther Keller, Richard Illi, Piotrek Madej und Justyna Wolinska, die mich auf meinen unzähligen Probenahmen bei Wind, Regen, Schneesturm, aber auch bei Sonnenschein auf den Greifensee begleitet haben. Die Erforschung von Ephippien aus dem Sediment war

- 113 - Danksagung zwangsläufig mit der Probenahme von Sedimentkernen verbunden - Piet Spaak, Hansruedi Bürgi, Piotrek Madej, Richard Illi und Christine Dambone danke für die gemeinsamen ‚Schlammschlachten’. Ein besonders spezieller Dank geht an Esther Keller und Regula Illi für das Auszählen all der quantitativen Zooplanktonproben. Ganz besonders möchte ich mich für die Hilfe von Colleen Durkin bei der Aufarbeitung und ihre Mithilfe bei der Analyse der vielen Daphnia-Proben von unserem 43 Seenprojekt bedanken.

Ich möchte mich natürlich auch bei all jenen bedanken, die mich in irgend einer Weise während den Probenahmen im Feld und der Laborarbeit unterstütz haben. Merci für euren Einsatz: Esther Keller, Richard Illi, Piotrek Madej, Christoph Tellenbach, Justyna Wolinska, Hansrudi Bürgi, Piet Spaak, Peek Keller, Astrid Löffler, Sandra Lass, Monika Winder, Mike Monaghan, Collen Durkin, Christine Dambone und alle weiteren HelferInnen.

Ich möchte mich bei Piet Spaak, Nelson Hairston, Katja Räsänen, Mike Monaghan, Chris Robinson, Andrew Park, und ganz speziell bei Justyna Wolinska, Christoph Tellenbach und Christian Rellstab für das Durchlesen, Kommentieren, Korrigieren und Ergänzen meiner Manuskripte bedanken!

Ein grosses Merci den UnterrichtsassistentInnen Verena Wenzelides, Stefan Aebischer, Gabi Meier Bürgisser, Kathi Turi-Nagy, Jacqueline Bernet, Janine Rüegg, Karin Gafner und Sarah Fässler für die gemeinsam verbrachte Unterrichtstätigkeit...

...dem Bibliotheksteam Monika Zemp, Lilo Schwarz, Jutta Studer und Bas den Brok für die tolle Zusammenarbeit...

...natürlich auch der gesamten Limnologie bzw. Aquatic Ecology für die nette und sehr lehrreiche Zeit...

...allen KollegInnen und FreundInnen, die ich während meiner langjährigen Pendelei im Zug zwischen Herzogenbuchsee und Stettbach kennen und schätzen gelernt habe: Andi, Judith, Dieter, Markus und Michelle. Das Zug- fahren war mit euch nie langweilig, und die schnellen Spurts auf die S3 am Morgen waren fast noch besser als der Morgenkaffee...

- 114 - Danksagung

...ganz speziell an Christoph Tellenbach für seine geteilte Begeisterung für finnischen Speed- und Power-Metal...

Natürlich hat nicht alles erst an der Eawag begonnen, ganz herzlich möchte ich mich bei meinen Eltern, meiner Oma und meinen Grosseltern bedanken, die mich für die Belange der Natur sensibilisierten und meine Begeisterung für alle Lebewesen geweckt haben. Paps ich zähle immer noch Daphnien und habe eine Million noch nicht ganz erreicht...

Herzlichen Dank meinem Bruder für seinen 24h-Super-Computer-Support!

Einen ganz speziell herzlichen Dank meinen Schwiegereltern, die immer für mich da sind und nie zögern Hilfe zu leisten.

Abschliessend ein SUPER GROSSES MERCI meinem Ehemann Peek, der mich in allen Belangen ununterbrochen unterstützte, all meine Eskapaden punkto Arbeitszeit tolerierte und der mich während meiner schlechten Phasen immer wieder motivierte weiterzumachen. Herzlichsten Dank Peek, dass du mich auch immer bei unserem gemeinsamen ‚Jogglen’ am Wochenende begleitet hast.

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