Avian Malaria and Interspecific Interactions in Ficedula Flycatchers

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1781

WILLIAM JONES

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0589-9 UPPSALA urn:nbn:se:uu:diva-377921 2019 Dissertation presented at Uppsala University to be publicly examined in Zootissalen, EBC, Villavägen 9, Uppsala, Friday, 26 April 2019 at 10:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Associate Professor Rebecca Safran (University of Colorado, Boulder).

Abstract Jones, W. 2019. Avian Malaria and Interspecific Interactions in Ficedula Flycatchers. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1781. 44 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0589-9.

Parasitism is a core theme in ecological and evolutionary studies. Despite this, there are still gaps in our knowledge regarding host-parasite interactions in nature. Furthermore, in an era of human-induced, global climatic and environmental change revealing the roles that parasites play in host life-histories, interspecific interactions and host distributions is of the utmost importance. In this thesis, I explore avian malaria parasites (haemosporidians) in two species of passerine birds: the collared flycatcher Ficedula albicollis and the pied flycatcher F. hypoleuca. In Paper I, I show that an increase in spring temperature has led to rapid divergence in breeding times for the two flycatcher species, with collared flycatchers breeding significantly earlier than pied flycatchers. This has facilitated regional coexistence through the build up of temporal isolation. In Paper II, I explore how malaria assemblages across the breeding ranges of collared and pied flycatchers vary. I find that pied flycatcher populations have significantly higher infection prevalence than collared flycatchers, but collared flycatchers have a higher diversity of parasites. Additionally, I find that recently colonised flycatchers have kept their original parasite assemblages while gaining further parasites from native pied flycatchers. In Paper III, I explore age-related patterns of malaria infections in collared flycatchers. I find that female collared flycatchers have higher overall infection rates than males and that infected female collared flycatchers have significantly higher mortality rates than uninfected females while males pay no survival cost. Despite this, female collared flycatchers do not pay a fitness cost, despite their shorter lifespans. In Paper IV, I explore nest defence behaviours of infected and uninfected collared flycatchers. I find that malaria infection significantly interacts with age and that young, infected collared flycatchers have a lower intensity of defence behaviours than uninfected individuals, while the opposite pattern is present in older collared flycatchers, with infected birds having higher defence behaviours. Therefore, I argue that Papers III and VI suggest patterns of terminal investment are present in collared flycatchers. Finally, in Paper V, I investigate parasite transmission in pied and collared flycatchers. I find that infected individuals of both species produce higher quantities of volatile organic compounds (VOCs) than uninfected individuals. Additionally, there is a significant increase in VOCs produced when the number of malaria gametocytes is higher. This suggests that malaria parasites are able to manipulate their hosts into producing insect-vector attracting compounds and that this is further increased at peak infectivity. These findings help to fill in some of the gaps in the literature regarding host-parasite relationships and the role of environmental change on hosts.

Keywords: ficedula, flycatcher, avian malaria

William Jones, Department of Ecology and Genetics, Animal ecology, Norbyvägen 18 D, Uppsala University, SE-752 36 Uppsala, Sweden.

ISSN 1651-6214 ISBN 978-91-513-0589-9 urn:nbn:se:uu:diva-377921 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-377921)

“An original idea? That can’t be too hard, the library must be full of them”

Stephen Fry

For Mum and Dad

4 List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Sirkiä, P.M., McFarlane, S.E., Jones, W., Wheatcroft, D., Ålund, M., Rybinski, J., & Qvarnström, A. (2018) Climate- driven build-up of temporal isolation within a recently formed avian hybrid zone. Evolution, 72(2):363–374 II Jones, W., Kulma, K., Bensch, S., Cichoń, M., Kerimov, A., Krist, M., Laaksonen, T., Moreno, J., Munclinger, P., Slater, F. M., Szöllősi, E., Visser, M.E. & Qvarnström, A. (2018) Inter- specific transfer of parasites following a range-shift in Ficedula flycatchers. Ecology and Evolution, 8(23):12183-12192 III Jones, W., Menon N.K. & Qvarnström, A. Sex-specific de- crease in longevity following malaria infection in a natural bird population. Submitted manuscript. IV Jones, W., da Cunha, C.S.G., Carra, L.G., Gallego-Abenza, M. & Qvarnström, A. Age and malaria infection affect nest defence behaviours in collared flycatchers. Submitted manuscript V Jones, W., Mozūraitis, R., McFarlane S.E., Emami, S.N. & Qvarnström, A. Malaria infected birds produce higher concen- trations of vector attracting compounds. Manuscript

Reprints were made with permission from the respective publishers.

Cover and other illustrations: Original artwork courtesy of Emma Wood.

6 Contents

Introduction ...... 9 Parasites, Pathogens and Parasitism ...... 9 Research Aims ...... 11 Materials and Methods ...... 12 Host species & study site ...... 12 Avian malaria ...... 14 Environmental variables (paper I) ...... 16 Malaria diagnosis protocol (papers II-V) ...... 16 Volatile collection and analysis (paper V) ...... 17 Results and Discussion ...... 20 Paper I Climate change drives temporal isolation in an avian hybrid-zone ...... 20 Paper II Broad-scale patterns of malaria infection in pied and collared flycatchers ...... 21 Paper III Sex-bias in infection rates and survival costs in collared flycatchers ...... 23 Paper IV Avian malaria, predators and terminal investment in collared flycatchers ...... 25 Paper V Malaria parasites manipulate avian hosts to be more attractive to insect vectors ...... 27 Conclusions and Future Perspectives ...... 30 Sammanfattning på Svenska ...... 32 Acknowledgements ...... 36 References ...... 39

7 Abbreviations

CF Collared flycatcher ERH Enemy release hypothesis GC-MS Gas chromatography – mass spectrometry LRS Lifetime reproductive success NMDS Non-metric multidimensional scaling NWH Novel weapon hypothesis PCR Polymerase chain reaction PF Pied flycatcher SPME Solid phase microextraction VOC Volatile organic compound

8 Introduction

“There seems to me too much misery in the world. I cannot persuade myself that a beneficent and omnipotent God would have designedly created the Ichneumonidae with the express intention of their feeding within the living bodies of caterpillars…”

So said Charles Darwin in a letter to the botanist Asa Gray in 1860 as he wrestled with his religious beliefs and scientific convictions. Yet Darwin was not the first person to try to understand the phenomenon that is parasitism (Suárez, 2018). Ancient Egyptians recorded roundworms and guin- eaworms in their papyrus scrolls and Aristotle described tapeworms from slaughtered animals (Cox, 2004). Yet it wasn’t until the mid-20th century that scientists such as John Haldane, Niko Tinbergen and Bill Hamilton began to look at parasites in the light of evolutionary biology and ecology (Foster, 1965). Since those days, the field of parasitology has spread to cover every aspect of parasite biology and the effects that they have on their hosts.

Parasites, Pathogens and Parasitism In an era of global environmental change, organisms are forced to adapt to new pressures such as climate change, biological invasions, changing food resources and parasites (Harvell et al., 2002; Parmesan & Yohe, 2003; Walther et al., 2002). Parasitism is thought to be the most common life- history trait and parasites and pathogens are ubiquitous in nature (Poulin, 2011; Schmid-Hempel, 2010). Parasites have overwhelmingly negative impacts on their hosts, including reduced reproductive performance (Knowles, Palinauskas, & Sheldon, 2010), survival (Ebert, Lipsitch, & Mangin, 2000) and competitive abilities (Park, 1948). Strategies can vary from endoparasit- ism, as utilised by many pathogens such as bacteria, viruses and protozoans where host cells or tissues are invaded to ectoparasitism, which is employed by organisms such as ticks, fleas or some fish which are external to host tissues. A slightly different form of parasitism occurs in the form of parasitoids that have a free-living stage but require a host to complete part of their life-cycle such as the ichneumon wasps. Parasitoids are different as killing the host is an obligatory part of their life-cycle, unlike traditional parasitism. Finally, there are the social parasites that exploit social structures of hosts.

9 The most famous examples of these include bird species that lay their eggs in other nests, such as cuckoos or whydahs (Schmid-Hempel, 2010). Parasites often have complex life-cycles which can include multiple hosts. This trait can be taken to extremes in some groups such as trematodes and cestodes. For instance, the trematode Halipegus occidualis requires four host species (frog, snail, ostracod and dragonfly) to complete its life-cycle (Esch, Barger, & Fellis, 2006). As a result, parasites have evolved complex ways to increase transmission success via host manipulation. Host manipulation can take many forms and can influence behaviour, life-history traits or phenotype to increase transmission success (Heil, 2016; Thomas, Poulin, & Brodeur, 2010). Some striking examples of host manipulation include the loss of fear or increase in risk-taking behaviours that are employed by Toxo- plasma spp. (Berdoy, Webster, & Mcdonald, 2000) or causing gigantism in freshwater snails (Guttel & Ben-Ami, 2014). Within species, parasites can have different effects on the sexes or age classes of their hosts. There is a general pattern for males to have higher infection prevalence due to antagonistic interactions between the immune system and sex hormones like testosterone (Clutton-Brock & Isvaran, 2007; Zuk & McKean, 1996). However, this is not an absolute rule and a few systems show female-biased parasite prevalence, usually due to specific idio- syncrasies in their behaviour (Klein, 2004; McCurdy, Shutler, Mullie, & Forbes, 1998). Meanwhile parasites can have specific effects and prevalence differences, depending on the age of the host. Finally, the role that global climate change will have on host-parasite interactions is a growing concern with hosts having different responses to parasites with fluctuating temperatures (Garvin, Abroe, Pedersen, Dunn, & Whittingham, 2006). Furthermore, as climate and environments change, host populations move and create novel assemblages, bringing novel parasites to naïve hosts (Cozzarolo, Jenkins, Toews, Brelsford, & Christe, 2018; Reullier, Pérez-Tris, Bensch, & Secondi, 2006; Scordato & Kardish, 2014). While presence of parasites can facilitate regional coexistence between species (Schall, 1992), they can also exacerbate species declines by effecting one species to a greater extent (Park, 1948). Understanding how hosts and parasites interact and how their ability to shift between host species is of ever-increasing importance beyond the fields of ecology and evolution. As emergence of new infectious diseases and zoonoses have direct impacts on human heath, crops and livestock (Cutler, Fooks, & Van Der Poel, 2010).

10 Research Aims This thesis explores the role that one specific group of parasites (avian malaria) has on intra- and interspecific interactions in two, closely related species of birds. To do this I first explore the role of climate change in mediating interspecific interactions and temporal isolation (Paper I). Then, I zoom out to look at geographic patterns and community ecology of malaria infection and explore how host population-shifts have facilitated parasite spillover (Paper II). Next, I explore the role that avian malaria has on survival and fitness (Paper III) and behaviour (Paper IV). Finally, I explore how parasites manage to manipulate their hosts to increase transmission success and how this can differ between closely related hosts (Paper V).

11 Materials and Methods

Host species & study site Collared and pied flycatchers Ficedula albicollis & F. hypoleuca are two widespread, hole-nesting passerines that breed across Europe in woodland habitats (figure 1). Pied flycatchers generally have a more northerly distribution, spreading from Great Britain to central Siberia and beyond the Arctic Circle in Scandinavia. There is also an isolated subspecies, F. h. iberiae, in the mountains of the Iberian Peninsula (Cramp, Perrins, Brooks, & Dunn, 1993; Lundberg & Alatalo, 1992). Collared flycatchers, by contrast have a more southerly and easterly distribution ranging from Germany to western Russia; the Italian Peninsula and the northern Balkans. In addition, there are isolated populations on the Swedish islands of Gotland and Öland and the Estonian island of Saaremaa, in the Baltic Sea (Cramp et al., 1993). There is a broad overlap in the distributions of the two species in central Europe and on the Baltic Islands. Hybridisation occurs throughout the zone of sympatry (Sætre, Kral, Bures, & Ims, 1999). Hybrids were thought to conform to Hal- dane’s rule with females being sterile and males being partially fertile; however recent evidence suggests that despite signals of past introgression (Sætre et al., 2001), male hybrids are also sterile (Ålund, Immler, Rice, & Qvarnström, 2013). Collared flycatchers are generally more competitive and more aggressive than pied flycatchers, and as a result, pied flycatchers have been slowly ex- cluded from the best quality habitats (Vallin, Rice, Arntsen, Kulma, & Qvarnström, 2012). However, pied flycatchers are able to deal with poor quality habitats (such as mixed or coniferous woodland) and colder and wet- ter environmental conditions, which allows for some regional coexistence on the island (Qvarnström, Wiley, Svedin, & Vallin, 2009; Rybinski et al., 2016).

12 Both species are migratory and migrate to woodland savanna and gallery forest habitats in sub-Saharan Africa during the non-breeding season (Curry- Lundahl, 1981; Salewski, Bairlein, & Leisler, 2002). Both species are entire- ly allopatric during this time with pied flycatchers migrating to West Africa and collared flycatchers migrating to south-central Africa. Breeding com- mences in late-April, when returning males compete for territories. Egg- laying occurs from mid-May with females of both species laying between 5- 8 eggs. Incubation is carried out solely by the female and takes approximately 18 days. After hatching, both parents feed the nestlings and after approximately 14 days the nestlings fledge.

Figure 1. Map showing the breeding distributions of pied (yellow) and collared flycatchers (blue). The overlap zone is denoted in green. The star indicates Öland and sampling locations for Paper II are marked with black circles.

Collared flycatchers first colonised northern Öland in the 1960’s and have subsequently spread throughout the island (Lundberg & Alatalo, 1992). Fly- catchers have been studied annually on Öland since 2002 in over 2000 nest boxes across 25 woodplots. Each bird is fitted with a unique alpha-numeric identifier code as a nestling or as an adult. Every individual is blood sampled, weighed and measured for a variety of different biometric measures including wing length, tarsus length and size of sexually selected traits such as forehead patch size and wing patch size. This thesis is mostly concerned with the populations on Öland. However, Paper II also involves sampling from other locations in Europe (see figure 1).

13 Avian malaria Avian malaria is a general term for the disease caused by three genera of haemosporidian parasites (Phylum: Apicomplexa). Of the three, Plasmodium is best known as its species are responsible for human malaria; however, Plasmodium species have been found in a wide range of vertebrate taxa including other mammals, birds, reptiles and amphibians. In addition, two other genera are widespread in avian hosts: Haemoproteus and Leucocytozo- on. The three genera have broadly similar life-cycles that involve an insect host and a vertebrate host. A wide variety of competent insect hosts have been described. Plasmodium spp. are most frequently transmitted by mosquitoes, particularly those belonging to the genus Culex. Haemoproteus spp. are transmitted by either Culicoides biting midges or louse flies of the family Hippoboscidae. Finally, Leucocytozoon spp. are most common in black flies from the family Simuliidae. Development is broadly similar in all three genera and in human malaria species. Briefly, sexual reproduction occurs in the gut of the insect vector and sporozoites migrate towards the salivary glands. From there they pass into the blood stream of the avian host where they migrate towards organs such as the liver. There they develop, proliferate and differentiate into merozoites. These merozoites are able to penetrate erythrocytes where they mature into gametocytes. When fully mature, the gametocytes burst from the erythrocytes into the blood plasma where they can be ingested by a new insect vector and this continue the cycle (Valkiūnas, 2005). As a group, haemosporidians have been found to have a mixed set of effects on their hosts. Some, such as Haemoproteus lineages have been histori- cally thought to be relatively benign in their hosts (LaPointe, Atkinson, & Samuel, 2012). However, they can have devastating effects on naïve host communities. For instance, avian malaria is thought to be one of the leading causes in population declines and extinctions of several species of Hawaiian honeycreepers (van Riper, van Riper, Goff, & Laird, 2006; Woodworth et al., 2005). Additionally, they have caused mass die-off of captive bird populations such as cranes (Jia et al., 2018). However, a growing body of research is discovering that these parasites do come with costs ranging from reduced fitness to higher mortality rates in a wide diversity of avian species (Knowles et al., 2010; Marzal et al., 2016). Haemosporidians as a group are both widespread and diverse. To date, roughly 3300 distinct lineages have been described from almost 1600 host species (Bensch, Hellgren, & Pérez-Tris, 2009). Many lineages are special- ists and are to a limited to one or two host species, however several generalist lineages are found in a wide variety of hosts. For example, the Plasmodi- um relictum lineage pSGS1 has been discovered in 126 species from 11 or- ders (Bensch et al., 2009). This choice of a generalist or specialist life-

14 Figure 2. Maximum-likelihood tree using a Tamara-Nei model of the 54 Haemo- proteus, Leucocytozoon and Plasmodium lineages found in collared and pied flycatchers. The tree was produced in Mega7 and based on a 480 bp fragment of the mitochondrial cytochrome b gene. Node markers indicate presence in pied flycatchers (black), collared flycatchers (white) or in both species (grey). Lineage names and sequences taken from the MalAvi database (Bensch, Hellgren, & Pé- rez-Tris, 2009). history strategy entails trade-offs for the parasite and it is a common trend to see a negative correlation between host diversity and prevalence in any given host species (Medeiros, Ellis, & Ricklefs, 2014). Generalists can find a suit- able host with greater ease; however, they also need to deal with a subsequently high variety of immunological threats from the host. A specialist, on the other hand can target their response to the immune system of their host (Hellgren, Pérez-Tris, & Bensch, 2009). A previous study on lineage diversity on Öland (Kulma, Low, Bensch, & Qvarnström, 2013) found 22 lineages in collared flycatchers and 14 lineages in pied flycatchers. The most common lineages are all Haemoproteus: H. majoris hPHSIB1 and H. pallidus hPFC1 in collared and pied flycatchers respectively. In addition, several

15 other lineages have been discovered in other populations of Ficedula flycatchers (figure 2).

Environmental variables (paper I) Spring temperature variables were downloaded from the Swedish Meteoro- logical and Hydrological Institute (SMHI) website. Data was collected from the monitoring station at the northern tip of Öland (Norra Udde). The best way to assess the progression of spring and the earliness of spring phenology is to use spring warmth sums (Charmantier et al., 2008). For this paper I used April warmth sums (with a base temperature of +5oC). This represents the bud-burst time of deciduous tree leaves. Seasonal food abundance varies dramatically across habitat types and timing of breeding for flycatchers is selected to match the peak of food abundance. Therefore, it was necessary to include a measure of habitat quality to account for this (Rybinski et al., 2016). To compare different habitat types with varying temporal abundances and availability of food I used the most common 11 tree species, covering the full gradient between high-quality deciduous forest and low-quality mixed forest. In addition, I assessed cater- pillar abundance associated with each tree species (hazel Corylus avellana, oak Quercus sp, ash Fraxinus excelsior, birch Betula sp, pine Pinus syl- vestris, alder Alna glutinosa, hornbeam Carpinus betulus, elm Ulmus glabra, lime Tilia cordata, spruce Picea abies and maple Acer sp) and then combined this estimate with nest-box point estimates of habitat tree species composition. Finally this point data was extrapolated to get habitat type estimates for the foraging range of each breeding flycatcher pair (Rybinski et al., 2016).

Malaria diagnosis protocol (papers II-V) 10-20µl of blood was collected from the brachial vein of each bird and stored either in 99% ethanol (2002-2013) or on Whatman FTATM filter paper cards (2013-2018). DNA was extracted from blood stored in ethanol using the high salt extraction method. Blood stored on filter paper was extracted using SDS/TRIS-guanidine thiocyanate (Smith & Burgoyne, 2004). Presence or absence of haemosporidians was assessed using nested polymerase chain reaction (PCR) protocols developed by Waldenström et al. and Hellgren et al (Hellgren, Waldenström, & Bensch, 2004; Waldenström, Bensch, Hasselquist, & Östman, 2004). For the first round of PCR, 1.0µl of DNA extract was amplified using the primers HAEMNF (5’CATATATTAAGAGAATTATGGAG-3’) and HAEMNR2 (5’AGAGGTGTAGCATATCTATCTAC-3’) which amplifies a 572 base- pair fragment of haemosporidian cytochrome-b gene, located on the mito- chondria of the parasite. 1.0µl of the resulting PCR product was then ampli-

16 fied again using a 479 base-pair fragment using the primers HAEMF (5’ATGGTGCTTTCGATATATGCATG-3’) and HAEMR2 (5’GCATTATCTGGATGTGATAATGGT-3’). 3µl of the resulting product was then stained with 2µl of GelGreen and inspected via gel electrophoresis on 1.5% agarose. The presence of a single band indicated haemosporidian infections. To reduce contamination risk DNA extraction and pre- and post- PCR steps were performed in different areas of the laboratory. In addition, negative (ddH20) and positive controls were run (1 negative control for every 11 samples; 1 positive control for every 40 samples). Positive samples were Sanger sequenced to ascertain lineage identity. Sequences were aligned using BioEdit software and compared with previously published sequences in the MalAvi database (Bensch et al., 2009). In paper V, concentration of gametocytes was assessed by visually in- specting blood smears. To create smears, a drop of blood was spread across a glass microscope slide and allowed to dry before fixing for 10 minutes in absolute methanol. Smears were then stained with a solution of Geimsa’s stain in phosphate buffer for approximately one hour. Gametocytes were counted using oil immersion light microscopy and a percentage of gametocytes was calculated from the number of parasite cells for every 1000 erythrocytes.

Volatile collection and analysis (paper V) Volatile organic compounds (VOCs) released from birds were collected via the dynamic aeration method (Millar & Sims, 1998). Each bird was placed in an individual 8x30cm, glass chamber (Sable Systems, Henderson, NV, USA), stoppered at each end and containing a metal grid at the base to allow comfort for the bird (see figure 3). The dry air was pushed into the chamber with a diaphragm vacuum pump (NMP 830 KNDCB; KNF Neuberger Inc., Freiburg, Germany) through an activated charcoal (Sigma-Aldrich Sweden AB, Stockholm, Sweden) at the flow rate of 0.6 L/min. One end of a collection trap (glass tube ID 5 mm x 6.0 cm) filled with 50 mg of 60/80 mesh activated Tenax TA absorbent (Sigma-Aldrich Sweden AB, Stockholm, Sweden) was attached to the outlet of a chamber and the other end of the trap was connected to another diaphragm vacuum pump pulling air at the rate of 0.55 L/min. By pulling out less air than supplying into the chamber, it ensured that no contaminated air from the laboratory would enter the system. VOCs from birds were sampled for 3 hours. Up to four chambers were used simultaneously to collect VOCs and at least one control sample per run was obtained by collecting VOCs from an empty chamber. Extraction was carried out in a climate cabinet set at 28oC to keep the flycatchers in their thermal neutral zone (Bushuev, Husby, Sternberg, & Grinkov, 2012). After the collection of VOCs, 250 μL of redistilled diethyl ether (Carlo Erba Reagents SAS, Val-de-Reuil, France)

17 was used to elute samples into glass vials which were closed with screw caps bearing Teflon septum. Samples were stored in a freezer and later transport- ed to the laboratory to for analysis. Prior to the analysis 30 ng of (Z)-10-tetradecen-1-yl acetate as an internal standard was added to the samples, afterwards samples were concentrated under a gentle flow of nitrogen and all content (approximately 1µl) was in- jected in a gas chromatograph.

Figure 3. Two male collared flycatchers in the VOC extraction chambers. Air is pumped over the birds from the right and simultaneously pulled through from the left. The analyses were performed using a HP 6890N gas chromatograph (GC) coupled with a HP 5973 mass spectrometer (MS) (Agilent Technologies INC, Santa Clara, CA, USA). The GC was equipped with a DB-5 capillary column (30 m x 0.25 mm x 0.25 µm; Agilent Technologies, Santa Clara, CA, USA). The injector and the detector temperatures were held isothermal at 230°C and 240°C, respectively. At the beginning of analysis, the GC oven temperature was held isothermal at 40°C for 2 minutes, then increased by 4°C at 7-minute intervals to 200°C, then 10°C each minute to 280°C and afterwards held isothermally at 280°C for 10 minutes. Helium was used as the carrier gas with a constant flow through the column at 1 mL min/min. Electron ionization mass spectra were determined at 70 eV and the ion source temperature held at 200oC. Relative amounts of the compounds in- jected were determined as areas under chromatographic peaks using comput- er processing software MSD Productivity ChemStation (v.02.01.1177) (Ag- ilent Technologies INC, Santa Clara, CA, USA). Compounds were identified by comparing their mass spectra and retention indexes with those present in the NIST-2008 MS library, afterwards with published retention index values

18 and finally with those of authentic standards. The retention index of the sep- arated compounds was calculated using the retention data from C8-C28 n- alkanes solution (Sigma-Aldrich Sweden) obtained under the same chromatographic parameters as those used for the analysis of volatile compounds. The chromatographic peaks of volatile fatty acids showed a certain degree of tailing on DB-5 capillary column, hence, the quantification of those compounds was carried out by displaying characteristic ion fragment m/z 60. Due to partial overlap of certain target chromatographic peaks which is a typical phenomenon of the samples constituting of a few hundred components, the following ion fragments were used for quantification: m/z 70 for heptanal; m/z 84 for octanal; m/z 93 for certain terpenes; m/z 108 for sul- catone.

19 Results and Discussion

Paper I Climate change drives temporal isolation in an avian hybrid-zone Human-induced climate change is causing rapid changes to the environment as is causing species’ ranges to shift at ever increasing rates, bringing species into secondary contact (Parmesan, 2006). In addition, organisms are experiencing phenological shifts to cope with a warming climate (Edwards & Richardson, 2004; Stevenson & Bryant, 2000). As organisms come into secondary contact, they need to develop mechanisms to avoid the fitness costs that are frequently associated with hybridisation (Ålund et al., 2013; Svedin, Wiley, Veen, Gustafsson, & Qvarnström, 2008). In this paper I explored the propensity for species to adapt to the novel pressure of a competitor. Specifically, I explored whether allochronic isolation (i.e., divergence in timing of breeding) has developed in pied and collared flycatchers on Öland. To do this I coupled climatic data and habitat quality measures with a long-term dataset of breeding collared and pied flycatchers (2002-2016) to assess breeding time, heterospecific pairing frequency and relative female fitness over time. I found that both species have responded to climate change by advancing their timing of breeding over just 14 years. However, selection on pied flycatchers to breed earlier was weaker leading pied flycatchers to generally breed later than collared flycatchers (figure 4). This has resulted in fewer interspecific pairings and thereby a reinforcement of reproductive isolation. This is interesting as it shows that even minor differences in the response to environmental change can quickly lead to the formation of allochronic isolation. Furthermore, these findings demonstrate the difficulty in making general predictions about the impact of climate change across populations, as even closely related species that occupy similar niches can react in different ways. Finally, it raises the prospect that despite the well documented negative impacts of global environmental change, in certain circumstances it might help to prevent local extinctions and facilitate regional cohabitation of closely related organisms.

Figure 4. Total number of breeding pairs of collared (left) and pied (right) flycatchers between 2002-2015 for each standardised laying date. Graphs are coupled with a smoothed function of relative fledging success across these standardised laying dates.

Paper II Broad-scale patterns of malaria infection in pied and collared flycatchers Two competing theories aim to predict what will happen when a host species colonises a new area: The Novel Weapon Hypothesis (NWH) and the Enemy Release Hypothesis (ERH). NWH predicts that the newly colonising species brings a suite of parasites with it that then disproportionately affect the native host species they infect. This has been illustrated in several systems including crayfish (Holdich & Reeve, 1991), squirrels (Rushton et al., 2006) and warblers (Reullier et al., 2006). Naïve local hosts are predicted to do worse in this scenario as they have not had a shared history with the parasite and therefore have not evolved the necessary immune responses. ERH predicts that the newly colonising species will escape its parasites in its core range and therefore will have a competitive advantage over the native host species. Evidence for this can be found in house sparrows, Passer domesti- cus, that were introduced to Peru and completely lost the avian malaria parasites from their natural range (Marzal et al., 2011). Previous work in collared and pied flycatchers identified high diversity of avian malaria lineages in both species, however collared flycatchers were shown to have a significantly lower prevalence (Kulma et al., 2013). There- fore, it was theorised that the system conformed to the ERH and that malaria infections play a role in the competitive asymmetry between the two species. However, a formal assessment of malaria diversity and prevalence away from Öland was needed to confirm this theory.

21 For this study, a total of 1503 birds were sampled from four populations of collared flycatchers (Czech Republic, Hungary, Poland and Sweden) and six populations of pied flycatchers (Finland, the Netherlands, Russia, Spain, Sweden and the United Kingdom) to assess parasite diversity and prevalence (See figure 1).

Figure 5. Boxplots showing (a) haemosporidian prevalence; (b) haemosporidian diversity (Shannon Index) and (c) haemosporidian lineage richness in pied (PF) and collared (CF) flycatchers

In total 5 Haemoproteus and 28 Plasmodium lineages were detected in across all populations. Flycatcher populations varied widely in their overall parasite community compositions with only three and four lineages being detected in all pied and collared flycatcher populations respectively (hPHSIB1, hCOLL2, hPFC1 and pRTSR1, pCOLL7, hCOLL2, hCOLL3) and only one lineage (hCOLL2) was found in both species and all seven populations. I found that pied flycatchers had a higher prevalence but a lower diversity of malarial lineages than collared flycatchers (figure 5). I also found that collared flycatchers in Sweden shared more lineages than allopatric populations. In addition, collared flycatchers had a higher prevalence and diversity of lineages transmitted in Africa (figure 6). This is interesting as it means that collared flycatchers do not conform to either the ERH, as they have not escaped any of their original parasites or the NWH as they have not transmitted any of their malaria lineages to pied flycatchers on Öland. Instead, we see that Swedish collared flycatchers gain parasite lineages from pied flycatchers when in sympatry. Pied flycatchers

22 could have higher overall infection rates than collared flycatchers due to potential immunological differences, with the two species using different strategies in the trade-off between tolerance and resistance (Råberg, Sim, & Read, 2007; Roy & Kirchner, 2000). I suggest that this explains the difference in infection rate between the species, while microhabitat differences explain the difference in diversity and prevalence between populations.

Figure 6. Non‐ metric multidimensional scaling (NMDS) plots of haemosporidian parasite communities (All infections, Haemoproteus, Plasmodium, African transmission, European transmission, and unknown transmission locations). Dark polygons represent pied flycatcher populations while pale polygons represent collared flycatcher populations.

Paper III Sex-bias in infection rates and survival costs in collared flycatchers A multitude of intrinsic and extrinsic effects can influence survival rates in organisms and these effects are not necessarily uniform across the sexes. For instance, there is a strong body of literature describing the role of intrinsic factors on survival and sex-biased mortality, such as the unguarded “X” hypothesis (or “Z” in ZW systems), antagonistic pleiotropy or the strength of sexual selection (Clutton-Brock & Isvaran, 2007; Maklakov & Lummaa, 2013; Tower, 2006; Trivers, 1985; Williams, 1957). However, many extrinsic factors also exert survival costs such as predation rates, climate change or parasitism (Acharya, 1995; Bize, Roulin, Tella, & Richner, 2005; Le Galliard, Fitze, Ferrière, & Clobert, 2005; Low, Arlt, Eggers, & Pärt, 2010).

23 The main aim Paper III was to see whether malaria infection has a negative effect on the survival of collared flycatchers and whether malaria infection has a negative effect on lifetime reproductive success (LRS). By looking at the infection status of 768 collared flycatchers of known age, I found that males and females not only have difference infection rates (43.1% vs 38.4%), but that infection rates vary between age classes. First year old males and females have the same prevalence of malaria parasites and then diverge at older age classes, with females peaking earlier (figure 7). First year birds are mostly displaying infections acquired in the previous year as nestlings. The higher infection rate for females in later age classes is likely explained by behavioural differences in the sexes, with females spending more time stationary when incubating eggs and hatchlings and therefore becoming easier targets for biting insects (Martínez-De La Puente et al., 2009).

Figure 7. Plot showing prevalence of avian malaria parasites across different breeding age classes of male (dashed line) and female (solid line) collared flycatchers. Birds that are five years old or older and grouped as ‘5+’. A Bayesian survival trajectory analysis (BaSTA) was performed on a subset of 244 collared flycatchers that were recruited to the population and bred in their first year, to investigate early life malaria infection on survival and mortality rates. This analysis showed that males and females had significantly different mortality curves, with males surviving longer than females. In addition, the analysis showed a significant effect of malaria on survival, but only for females. Male survival was not significantly affected by malaria infection (figure 8). This might be explained by differential allocation and investment of resources by the sexes. Females invest heavily into egg pro-

24 duction and incubation whereas males only contribute to nest defence and nestling provisioning (Cichoń, 2000). Finally, LRS was unaffected by malaria infection. This is interesting, as it suggests that despite their shorter lifespan, infected female collared flycatchers have the same reproductive output as uninfected individuals. This suggests that infected individuals are boosting their reproductive effort and trading-off future reproduction for current reproduction. Therefore, infected female collared flycatchers are engaging in terminal investment to maintain fitness.

Figure 8. Mortality and survival curves with 95% confidence intervals for each category (male uninfected, male infected, female uninfected, female infected), fitted by a Weibull mortality model. Plots b0 and b1 show posterior distributions of the model parameters. b0 represents the shape parameter and b1 is the scale parameter.

Paper IV Avian malaria, predators and terminal investment in collared flycatchers Defending a nest from predators can be costly in a variety of ways and parents need to weigh up the risks and benefits of defending their offspring from attack (Montgomerie & Weatherhead, 1988). As a result, birds should adjust their investment into nest defence depending on their age, with older birds engaging in riskier behaviour due to the terminal investment hypothesis (Trivers, 1972). In addition, individuals that are infected with parasites need to do a similar trade-off due to potentially shortened lifespans (Nordling, Andersson, Zohari, & Gustafsson, 1998; Stjernman, Råberg, & Nilsson, 2004). On Öland, collared flycatchers are exposed to a variety of predators. Some, like Eurasian sparrowhawks Accipiter nisus predominantly target adult birds and fledglings. Others like great spotted woodpeckers

25 Dendrocopos major and pine martens Martes martes target nestlings or incubating females (Post & Götmark, 2006; Sonerud, 1985; Wesołowski, 2002). In this paper, I aimed to test the effects of age and malaria infection on nest defence behaviours. To investigate this, a taxidermy model of a sparrowhawk was placed on the nest box of 103 collared flycatcher pairs when nestlings were between 8 and 10 days old. Over a period of 10 minutes, an observer scored the frequency of a number of anti-predator behaviours such as alarm call rate, wing flicking and minimum and average distance to the predator.

Figure 9. Minimum distance to the predator for male and female collared flycatchers during the predator presentation trial. We see that males got significantly closer to the predator than females. I found that male and female collared flycatchers differ in the distance that they are willing to approach a predator with males getting closer than females, however malaria infection had no significant effect on this behaviour (figure 9). Furthermore, I found that there was a significant interaction between age and malaria on nest defence behaviours. One-year old individuals that were infected with malaria flicked their wings and alarmed at a lower frequency than uninfected individuals; meanwhile the opposite happened with older individuals, where older individuals that were infected engaged in a higher intensity of defence behaviours.

26 When I compared uninfected older and first year birds I found a significantly higher intensity of defence behaviours. This could be due to experi- ence of the birds. Other systems have shown similar patterns where young, naïve individuals alarm more than older birds (Wiebe, 2004). The difference between infected old and young birds could be due to parasite infection stage with a greater proportion of younger birds experiencing recent infection and therefore a higher infection intensity (Sol, Jovani, & Torres, 2003).

Figure 10. Minimum distance to the predator for male and female collared flycatchers during the predator presentation trial. We see that males got significantly closer to the predator than females. This suggests evidence for terminal investment in collared flycatchers, expressed in their behaviour. With older, infected individuals trading-off future reproduction for current reproduction. This finding is complimented by those in paper III, where a survival cost of malaria infection is demon- strated.

Paper V Malaria parasites manipulate avian hosts to be more attractive to insect vectors

27 Figure 11. Relationship between gametocytaemia and quantity of VOCs produced in pied (A) and collared flycatchers (B). There is a significant relationship between the concentration of VOCs and gametocytaemia. Host-parasite interactions are some of the most dynamic processes in nature. Parasites often have complex life-cycles involving multiple hosts and need to employ a variety of methods to increase their chance of successful transmission between host species. Haemosporidians use female biting insects such as mosquitoes or Culicoides midges as a vector and for their own sexual reproduction (Valkiūnas, Liutkevičius, & Iezhova, 2002). These insects find their blood-meals by using a complex set of visual and chemical cues. These chemicals or VOCs include a cocktail of aldehydes, monoterpenes, alcohols and other aromatic compounds that are mostly by-products of metabolic processes of the host (Logan et al., 2008; Martínez-De La Puente et al., 2009). Previous studies have shown that humans that are infected with malaria are more attractive to insect vectors and recent evidence suggests that haemosporidians are able to manipulate the host’s metabolism to increase production of these compounds and thereby making infected individuals more attractive (Emami et al., 2017). Additionally, there is some evi-

28 dence that higher prevalence of gametocytes in a host (gametocytaemia) increases the attractiveness of the host to malaria vectors (Day & Edman, 1983). Yet the link between VOC production and gametocytaemia has yet to be made (Busula, Verhulst, Bousema, Takken, & de Boer, 2017). In this study I investigated whether collared and pied flycatchers produced similar compounds and whether their production was increased in infected birds. In addition, I hypothesised that higher gametocytaemia levels (i.e. the number of the sexually mature gametocytes) should cause VOC production to be even higher as gametocytes are the stage that requires a switch in host. Of 47 relevant compounds produced, I found that three of them (1- octanol, nonanal and decanal) were produced at higher quantities in infected individuals, furthermore we see that production of 1-octanol and nonanal correlated with gametocytaemia (figure 11). Finally, I found a significant difference in the production of compounds between pied and collared flycatchers, with pied flycatchers producing higher overall quantities of 1- octanol and nonanal (figure 12). This is a particularly exciting finding as it is the first to link gametocyte levels to production of VOCs. Furthermore, it could explain the higher malaria prevalence observed on Öland and in the broader range of pied flycatchers. To my knowledge, this is the first study finding a correlation gametocyte intensity and VOC production. The discovery of this phenomenon will great- ly aid future research on malaria transmission.

Figure 12. Abundance of VOCs in pied and collared flycatchers for 1-octanol (left) and nonanal (right). Production of both compounds is higher in pied flycatchers.

29 Conclusions and Future Perspectives

This thesis has focused on intra- and interspecific interactions in Ficedula flycatchers from how climate change has facilitated regional cohabitation via the development of temporal isolation to species and sex-specific effects of parasite infection. By using climatic, habitat and breeding data from collared and pied flycatchers in Paper I I discovered that collared flycatchers have advanced their mean breeding time in response to climate change faster than pied flycatchers. This finding has added some complexity to the general trend of the negative effects of climate change. From there I focused on haemosporidian parasites, their diversity and the costs that hosts bear when infected. In Paper II I find that malaria infection varies substantially in diversity and prevalence across the breeding distributions of collared and pied flycatchers. Collared flycatchers have a lower overall prevalence, but a higher diversity of lineages than pied flycatchers. In addition, collared flycatchers on Öland are more similar in their parasite community composition to pied flycatchers, suggesting an asymmetrical transfer of parasites from pied to collared flycatchers. These findings show that the Novel Weapon Hypoth- esis and the Enemy Release Hypothesis are both flawed in this system with the more competitive and newly collared flycatchers gaining parasites from the less competitive pied flycatchers. From there I move from spatial to temporal differences in malaria infection. In Paper III I find that malaria infection varies with and sex, with female collared flycatchers having higher overall infection rates and have a greater chance of infection over their lifespan than males. Furthermore, I found that avian malaria does not have an effect on the lifetime reproductive success of collared flycatchers, but it does come with a survival cost for females. In Paper IV I delve further into the impacts of these parasites on collared flycatchers by showing that malaria infection interacts strongly with age in nest-defence behaviours. Young, infected individuals engage in lower intensity displays than uninfected individuals, while the opposite pattern holds for older birds. Therefore, Papers III and IV show that collared flycatchers are able to make a trade-off between current and future reproduction and also terminally invest in reproduction when infected. Finally, in Paper V I find that malaria parasites success- fully manipulate their hosts to produce higher quantities of vector-attracting compounds and that these compounds are produced at higher quantities with higher gametocytaemia.

30 Many of these findings would not be possible without the utilisation of a long-term study system. In addition, this thesis demonstrates the versatility of Ficedula flycatchers for ecological studies. Papers I, III and IV make full use of the 16 years of data that has been accumulated in the flycatcher system on Öland thus far and have given insights into the fields of speciation while also providing a rare opportunity to couple malaria infections with host traits such as age and accurate measures of lifetime reproductive success. Paper II highlights the importance of investigating ecological factors across a broad spatial scale. Meanwhile, Paper V has offered novel insights into host manipulation, and may help to explain why ecologically similar species can vary in parasite prevalence. Despite this, we still know relatively little about the behaviour and ecology of pied flycatchers during the non-breeding season and next to nothing about collared flycatchers. Above all, this lack of knowledge is due to the logistic difficulties of studying these birds in Africa, particularly collared flycatchers which appear to confine themselves to politically unstable and climatically problematic parts of the continent. Yet to gain a better understanding of parasite transmission and survival, field studies in these regions would be highly beneficial. This thesis highlights several future research paths in this field. Firstly, species are able to rapidly isolate themselves from hybridisation risk, yet how broad this ability is should be a priority for researchers interested in the impact of climate change on speciation and hybrid-zones. Secondly, a greater understanding of the community ecology of avian malaria lineages would help understand parasite turnover, spillover and transmission probability. This information is crucial for understanding how new lineages and parasites emerge and spread which is important information in an era of rapid environmental change. Secondly, this thesis sheds light on an under researched field in haemosporidian studies: host manipulation. How malaria parasites can increase their transmission success and how much variation there is in their ability to do this has implications beyond the realms of ecology and evolution.

31 Sammanfattning på Svenska

Parasitism är ett vanligt sätt att leva på i naturen och används som livsstra- tegi av ett brett spektrum av organismer - från virus till evertebrater. En parasit utnyttjar sin värd för egen vinning på ett sätt som skadar värdorgan- ismen. Även investering i försvarsmekanismer, såsom immunförsvaret, kan innebära en ökad kostnad för värdorganismen som måste hushålla med sina begränsade resurser. Parasitens strävan efter att utnyttja värdorganismens resurser för sin egen fortplantning och värdorganismens strävan att försvara sig leder till en ständig pågående evolutionär kapplöpning. Parasiter, med sina korta generationstider, har ofta en fördel i den här kapprustningen och kan orsaka akuta hot, inte bara för vilda populationer av växter och djur, utan även för oss människor. Att förstå hur parasiter och värddjur interagerar med varandra och hur den evolutionära kapprustningen mellan dem går till har därför länge varit ett centralt forskningsområde inom ekologi och evolut- ionsbiologi. Min avhandling fokuserar på förekomst, diversitet och effekter av fågel- malaria hos svartvit flugsnappare Ficedula hypoleuca och halsbandsflug- snappare F. albicollis. Flugsnappare är skogslevande småfåglar som före- kommer i Europa. Svartvitflugsnappare förkommer häckande i mer nordliga och västliga delar av Europa medan halsbandsflugsnapparen har en mer syd- lig och östlig utbredning. Det finns två kontaktzoner där båda arterna före- kommer och hybridiserar med varandra: en bred hybridzon i centrala Europa och en hybridzon på Öland och Gotland i Sverige. Halsbandsflugsnapparen är mer aggressiv och dominant i revirstrider vilket leder till att svartvita flugsnappare drivs iväg från de föredragna habitaten av lövskog i regioner där båda arterna förekommer. Båda arterna flyttar till Afrika under vintern men använder olika flyttvägar och övervintringsområden i Afrika. Jag har valt att studera de här båda värdarterna och fågelmalaria för att kunna undersöka några av de mest centrala frågorna inom parasitologin. Några av de mest brådskande frågeställningarna inom ekologi och evolut- ionsbiologi handlar om att mäta och förutsäga vilka effekter mänsklig påver- kan på klimat och miljö har på levande organismer. Global uppvärmning, habitatförlust, habitatfragmentering och införsel av exotiska arter ger upphov till nya artsammansättningar. När värdorganismer ändrar sina utbrednings- områden flyttar deras parasiter med dem vilket leder till exponering av nya, naiva värdar. Klimatförändringar kan även leda till att värdarna får försäm- rad förmåga att försvara sig från parasiter som de har exponerats för tidigare.

32 Förståelsen för samspelet mellan parasiter och deras värdar samt parasiters förmåga att kolonisera nya värdorganismer blir därför allt mer viktigt. Detta eftersom uppkomsten av nya sjukdomar och zoonoser kan ha långtgående effekter på människor, djurs och växters hälsa. I min avhandlings första kapitel undersöker jag hur den pågående globala klimatförändringen påverkar hybridzonsdynamiken hos flugsnapparna. Generellt sett gör de pågående klimatförändringarna att unga arter som har varit separerade i tiotusentalsår kommer i kontakt med varandra i allt snabb- bare takt. Halsbandsflugsnapparen tillhör en av arterna som förutspås vidga sitt häckningsområde norrut. Dessutom gör stigande vårtemperaturer att många växter och djurs fortplantningscykler tidigareläggs. Detta är speciellt viktigt för skogslevande, insektsätande fåglar som är beroende av att anpassa sin häckning efter tillgången på fjärilslarver som de behöver för att mata sina ungar med. Jag fann att temperaturen under våren har ökat under de senaste fjorton åren på Öland och att båda flugsnapparartena har svarat genom att tidigarelägga sin häckning. Dock har halsbandsflugsnapparen svarat starkare på förändringen i temperatur vilket i sin tur har gjort att arterna har blivit mer separerade i häckningstid. Den här studien visar att klimatförändringar kan ha olika effekter även på väldigt snarlika arter och att sådana skillnader kan gynna regional samexistens. I det här fallet minskar risken för konkur- rens och hybridisering mellan de olika flugsnappararterna eftersom de delvis häckar i olika tidsfönster under våren och försommaren. I min avhandlings andra kapitel undersöker jag mönster av förekomst och diversitet av fågelmalaria över stora delar av flugsnapparnas häcknings- utbredning. Den här typen av storskaliga studier är viktiga av flera olika skäl. För det första är parasitdiversitet över en värdorganisms utbrednings- område en viktig ledtråd för att förstå hur värdorganismen påverkas av sina parasiter. I en era av globala miljöförändringar som leder till förändringar i många arters utbredning är det också troligt att närbesläktade arter utbyter parasiter sinsemellan. Två olika teorier förespår att invasiva arter som snabbt utökar sin utbredning kommer att ha en fördel i det här sammanhanget. ”Be- frielse från fienden”-hypotesen förutspår en konkurrensfördel hos invasiva arter som sprider sig till nya områden genom att de lämnar sina parasiter bakom sig. ”Nya vapen”-hypotesen däremot förutspår att invasiva arter har en konkurrensfördel över lokala arter genom att ta med sig nya parasiter som de lokala arterna saknar ett effektivt försvar mot. Jag fann att halsbandsflug- snappare hade lägre generell prevalens av malaria men en högre diversitet av olika typer av fågelmalaria i jämförelse med svartvit flugsnappare. Dessutom hade halsbandsflugsnapparna som häckar i den nyligen koloniserade region- en i Sverige troligtvis plockat upp parasiter från de lokala svartvitflugsnap- parna. Trots att halsbandsflugsnapparna lyckas konkurrera ut svartvit flugsnappare, så kan det inte förklaras av någon av de två hypoteserna. En högre genetisk diversitet av fågelmalaria hos halsbandsflugsnapparen kan också delvis förklaras av genetiska varianter av malaria som sprids under vintern i

33 Afrika. I regioner där halsbandsflugsnapparen övervintrar är det regnperi- oder vilket sammanfaller med större förekomst av myggor och andra vektorer som sprider fågelmalaria. Det är möjligt att en högre exponering av olika typer av malaria har selekterat fram ett bättre immunförsvar hos halsbandsflugsnapparen som förklarar den lägre prevalensen av malaria. Parasiter kan ha olika effekter också mellan individer inom samma art till exempel beroende av kön och ålder. Hanar förväntas ofta ha högre prevalens av parasiter på grund av antagonistiska interaktioner mellan immunsystemet och könshormoner som testosteron. Att förstå variation i hur individer på- verkas av parasiter är viktigt både på kort sikt när man vill förstå hur en population påverkas samt på lång sikt för att förespå den evolutionära kapprustningen mellan parasit och värd. I kapitel III och IV undersöker jag där- för denna typ av variation i parasitprevalens och i effekter på värddjuret. I kapitel III fann jag att halsbandsflugsnapparhanar och honor hade signifikant olika prevalens av malaria. Honorna var oftare infekterade av malaria men först efter första häckningssäsongen vilket tyder på att det är en skillnad i reproduktivt beteende som driver den högre risken att drabbas av malaria hos honor. En tänkbar förklaring är att honorna är relativt mer exponerade för insektsvektorer när de ligger stilla och ruvar sina ägg. Jag fann även att honor betalade en högre överlevnadskostnad som följd av malariainfektion. Den högre infektionsrisken och kostnaden av malaria bidrar till att honor har en signifikant kortare livslängd än hanar i populationen jag studerar på Öland. I kapitel IV, fann jag att infektion av malaria och ålder påverkade fåglar- nas försvarsbeteende mot en bopredator. Genom att placera en uppstoppad sparvhök på holken fann jag att fåglar som var minst två år gamla uppvisade mer intensiva boförvarsbeteende (varningsläten och vingslag) när de var infekterade med malaria än när de inte var infekterade. Eftersom malariainfektion leder till ökad mortalitet är en tänkbar förklaring att avvägningen mellan nuvarande och framtida fortplantning skiljer sig mellan dessa båda grupper av individer. En malariainfekterad fågel har sämre framtidsutsikter och är kanske därför villig att ta större risker för att förvara sina nuvarande ungar (så kallad terminal investering). Hos individer som häckade för fösta gången fann jag ett omvänt mönster. En tänkbar förklaring kan vara att yngre individer drabbas hårdare av akuta infektioner och därför kan vara i sämre kondition. Det vill säga att dessa unga infekterade individer inte är mindre villiga att uppoffra sig för sina ungar de har helt enkelt inte tillgång till resurser för att göra det på grund av att de är nedsatta av sjukdomen. Parasiter har ofta en komplex livscykel som kan inkludera flera olika vär- dar. I extrema fall kan det förekomma parasiter som behöver upp till fyra olika värdorganismer för att slutföra sin livscykel. Som ett resultat av detta behöver parasiter utveckla komplexa sätt att öka sin spridning på vilket kan involvera olika sätt att manipulera värden på. Värdmanipulation kan ta många olika uttryck och ge upphov till förändringar i värdens beteende eller

34 livscykel som gynnar parasitens spridning. Ett slående exempel av värdma- nipulation inkluderar ökad benägenhet till risktagande som orsakas av para- siten Toxoplasma spp. där gnagare förlorar sin rädsla för katter och istället attraheras av lukten av katturin. I kapitel V undersöker jag hur malariaparasiter påverkar sina värdar för att öka sin egen spridning. Genom att kvantifiera mängden av volatila orga- niska ämnen som avges från flugsnappare kunde jag visa att infekterade individer avger större mängder av ämnen som attraherar vektorer i luften som omger dem. Dessutom hittade jag ett positivt samband mellan mängden malaria gametocyter (spridningsformen av malaria) uppmätta i blodutstryk från individer och mängden av ämnen som attraherar vektorer som spreds i luften kring dem. Detta är en fascinerande upptäckt som tyder på att malari- aparasiten kan öka sannolikheten för att spridas just när den har en infekt- ionstopp i värden. Min avhandling har presenterat en rad nya upptäckter som ligger helt i linje med centrala teorier inom ekologi och evolutionsteori, som t. ex. före- komsten av en terminal investering. Mina resultat har också visat på en större komplexitet i samband med byte av värd än som förutsägs av de två vedertagna hypoteserna ”befrielse från fienden” eller ”nya vapen”. Dessutom har jag upptäckt ett nytt samband mellan en värds attraktivitet för vektorer och stadium av malariainfektion som har implikationer som potentiellt sträcker sig långt utanför mitt eget forskningsfält.

Translation by Anna Qvarnström

35 Acknowledgements

My first words of thanks must go to the tens of thousands of Collared and Pied Flycatchers that have been used to various degrees in the making of this thesis. A rough calculation suggests that I have personally processed about 1650 since joining the project, a small fraction of the individuals that have been used over the years! At every step of the way I have kept ethical considerations in mind and I can only hope that their exploitation has been of use beyond this thesis. Ethical permissions were granted under licence by the Swedish Ringing Authority and the Swedish Animal Ethics Board. This thesis has been funded primarily by Vetenskapsrådet, but also by the Bjur- zon travel grant, the Liljewalch travel grant, Stiftelsen för Zoologisk For- skning and the Royal Swedish Academy of Sciences.

A single person cannot write a thesis without the help and support of friends, family and colleagues and this thesis is no exception. So many people have helped me on this path and not least among them has been Anna. Thank you so much for all the guidance and mentoring throughout this process. You have an uncanny knack of being able to see the value and give clarity to every piece of work and scrap of data when I’ve felt lost. At the same time, you’ve created a safe, welcoming and most importantly – exciting lab and it has been an absolute pleasure to be part of it. I couldn’t have asked for a better supervisor. Huge thanks also go to Staffan, my second supervisor and malaria guru. Thank you for agreeing to supervise me before you even met me! Special thanks also need to go to David B. and Katja for making an excellent PhD committee tag team. Thank you to Mats for welcoming me to Animal Ecology, Göran, for your leadership and to Ingrid for your help and support, you always have our best interests at heart and always have our backs.

The guys at Lund University have been so important in helping me get my head around everything malaria, especially Elin and Xi, thank you for all the memories and beers drunk around the world.

My project has involved a lot of time in the lab, thank you so much to Reija and Gunilla for your time and patience and for answering all my silly ques- tions. Thank you, Kevin, Peter and Yvonne for all the interesting discussions about malaria and flycatchers and your help in the lab. Thank you too

36 to all the co-authors of the chapters for absolutely everything that you’ve done.

To Claus, Anssi, Martin, Frank, Per and all the other birders at the EBC, thank you for taking me out to see all the fantastic birds Sweden has to offer, from racing against the light to see the Caspian Plover to surviving Caper- caillie attacks. To Mattias, my long-suffering office mate, thank you for putting up with the chaos that was my desk and the ever-encroaching field equipment and samples. Patrik, you were one of the first people I met in Sweden when we started out master’s together and then our PhD’s on the same day; you are truly an analyst and a therapist. Thank you for all the good times with Jordi, Marloes, Deike, Carla, Marta, Gonçalo, Johanna S., Basil, Kerstin and the rest. Paula, you are a true original. Thank you for being my opposite in so many ways; never stop being an iconoclast. Josefine you have been a rock for me, thank you so much for our mutual grumbling sessions! Sara, you are one of the loveliest people I know, and you are an inspiration, keep on shining! Jaelle, somehow it feels like you’ve been here from the start. Our chats have always cheered me up, let’s do our Aussie roadtrip! Johanna L-R., You always have a kind word to say and you’re a daemon at fyra i rad! Warren, Anders, Foteini, Elina, Helen, Kai, Maria, Rado, Karo, Masahito, Karl, Brian, Javi, Ivain, Germán, Elisabeth, David O., Foteini, Zuzana, André, Julian, Tom, Martyna, Jacob, Mette, Steve, Richard, Pablo, Lars and everyone else at the department, past and present, for fikas, seminars, cocktails and discussions over the years. You all give Animal Ecology a fantastic vibe.

To Bo, thank you for all the great memories in Uppsala, Stockholm and beyond and for introducing me to schlager and Midori! Hernán, the best of friends can be made at the most unlikely times. Emma, thank you for the wonderful illustrations and vignettes for this thesis. Erik and Grace, you arrived near the end of my PhD journey, but you’ve made a big impact. I’ve enjoyed our birding forays immensely. Jack, thank you for ever and always being uniquely you.

One of the great strengths of the Qvarnström Lab are the close relationships we have. The work would not have been possible without the hard work and dedication of many field assistants and students over time. There are too many to name, but I must single out Mario, Joana, Matthew, Tiago and Fraser for going above and beyond the call of duty on numerous occasions. Extra special thanks go to the many landowners on Öland, particularly Yvonne and Bosse, who have not only provided us accommodation and allowed us to have nest-boxes on their property but have also been so gener- ous with their time and spirit, Tack så jätte mycket, jag kommer aldrig att glömma mina dagar på Öland. Thank you to Kasia for supervising me as a

37 master’s student and for setting me on this road. David W., thank you so much for your mentorship in and out of the field. Thanks for making field days so much lighter! Kuba and Päivi and for your stimulating discussions and for sharing long days in the field. Caro, good luck for the future, I’m so excited to see the direction your research will go in!

Eryn and Murielle, my flycatcher family! It’s hard to put into words exactly how much your friendship and guidance has meant to me from the very beginning. You are true inspirations, and I am in constant awe of you both. You’ve guided me through so much of this process and I’m looking forward to future work and fun with you guys in the years to come.

To my parents, Nick and Jane, my sister, Alice and my grandparents. From the earliest days, you’ve taken the fact that there’s a crazy bird guy in the family in your stride. Thank you for not only accepting that, but also for nurturing and encouraging me at every turn. Finally, to Javier, what else is left to say other than, quiérote.

38 References

Acharya, L. (1995). Sex-biased predation on moths by insectivorous bats. Animal Behaviour, 49(6), 1461–1468. https://doi.org/10.1016/0003-3472(95)90067-5 Ålund, M., Immler, S., Rice, A. M., & Qvarnström, A. (2013). Low fertility of wild hybrid male flycatchers despite recent divergence. Biology Letters, 9, 20130169. https://doi.org/10.1098/rsbl.2013.0169 Bensch, S., Hellgren, O., & Pérez-Tris, J. (2009). MalAvi: A public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. Molecular Ecology Resources, 9(5), 1353–1358. https://doi.org/10.1111/j.1755-0998.2009.02692.x Berdoy, M., Webster, J. P., & Mcdonald, D. W. (2000). Fatal attraction in rats infected with Toxoplasma gondii. Proceedings of the Royal Society B: Biological Sciences, 267(1452), 1591–1594. https://doi.org/10.1098/rspb.2000.1182 Bize, P., Roulin, A., Tella, J. L., & Richner, H. (2005). Female-biased mortality in experimentally parasitized alpine swift Apus melba nestlings. Functional Ecology, 19(3), 405–413. https://doi.org/10.1111/j.1365-2435.2005.00995.x Bushuev, A. V., Husby, A., Sternberg, H., & Grinkov, V. G. (2012). Quantitative genetics of basal metabolic rate and body mass in free-living pied flycatchers. Journal of Zoology, 288(4), 245–251. https://doi.org/10.1111/j.1469- 7998.2012.00947.x Busula, A. O., Verhulst, N. O., Bousema, T., Takken, W., & de Boer, J. G. (2017). Mechanisms of Plasmodium-enhanced attraction of mosquito vectors. Trends in Parasitology. https://doi.org/10.1016/j.pt.2017.08.010 Charmantier, A., McCleery, R. H., Cole, L. R., Perrins, C., Kruuk, L. E. B., & Sheldon, B. C. (2008). Adaptive phenotypic plasticity in response to climate change in a wild bird population. Science, 320(5877), 800–803. https://doi.org/10.1126/science.1157174 Cichoń, M. (2000). Costs of incubation and immunocompetence in the collared flycatcher. Oecologia, 125(3), 453–457. https://doi.org/10.1007/s004420000461 Clutton-Brock, T. H., & Isvaran, K. (2007). Sex differences in ageing in natural populations of vertebrates. Proceedings of the Royal Society B: Biological Sciences, 274(1629), 3097–3104. https://doi.org/10.1098/rspb.2007.1138 Cox, F. E. G. (2004, June). History of human parasitic diseases. Infectious Disease Clinics of North America. https://doi.org/10.1016/j.idc.2004.01.001 Cozzarolo, C.-S., Jenkins, T., Toews, D. P. L., Brelsford, A., & Christe, P. (2018). Prevalence and diversity of haemosporidian parasites in the yellow-rumped warbler hybrid zone. Ecology and Evolution, (1), 1–14. https://doi.org/10.1002/ece3.4469 Cramp, S., Perrins, C. M., Brooks, D. J., & Dunn, E. (1993). Handbook of the birds of Europe, the Middle East and North Africa : the birds of the Western Palearctic. Volume VII, Flycatchers to Shrikes. Oxford: Oxford University Press.

39 Curry-Lundahl, K. (1981). Bird migration in Africa, Vols 1 & 2. London: Academic Press. Cutler, S. J., Fooks, A. R., & Van Der Poel, W. H. M. (2010). Public health threat of new, reemerging, and neglected zoonoses in the industrialized world. Emerging Infectious Diseases. https://doi.org/10.3201/eid1601.081467 Day, J. F., & Edman, J. D. (1983). Malaria renders mice susceptible to mosquito feeding when gametocytes are most infective. The Journal of Parasitology, 69(1), 163. https://doi.org/10.2307/3281292 Ebert, D., Lipsitch, M., & Mangin, K. L. (2000). The effect of parasites on host population density and extinction: experimental epidemiology with Daphnia and six microparasites. The American Naturalist, 156(5), 459–477. https://doi.org/10.1086/303404 Edwards, M., & Richardson, A. J. (2004). Impact of climate change on marine pelagic phenology and trophic mismatch. Nature, 430(7002), 881–884. https://doi.org/10.1038/nature02808 Emami, S. N., Lindberg, B. G., Hua, S., Hill, S. R., Mozuraitis, R., Lehmann, P., … Faye, I. (2017). A key malaria metabolite modulates vector blood seeking, feeding, and susceptibility to infection. Science, 355(6329), 1076–1080. https://doi.org/10.1126/science.aah4563 Esch, G. W., Barger, M. A., & Fellis, K. J. (2006). The transmission of digenetic trematodes: Style, elegance, complexity. Integrative and Comparative Biology, 42(2), 304–312. https://doi.org/10.1093/icb/42.2.304 Foster, W. D. (1965). A history of parasitology. Edinburgh (& London): E. & S. Livingstone. Garvin, J. C., Abroe, B., Pedersen, M. C., Dunn, P. O., & Whittingham, L. A. (2006). Immune response of nestling warblers varies with extra-pair paternity and temperature. Molecular Ecology, 15(12), 3833–3840. https://doi.org/10.1111/j.1365-294X.2006.03042.x Guttel, Y., & Ben-Ami, F. (2014). The maintenance of hybrids by parasitism in a freshwater snail. International Journal for Parasitology, 44(13), 1001–1008. https://doi.org/10.1016/j.ijpara.2014.06.011 Harvell, C. D., Mitchell, C. E., Ward, J. R., Altizer, S., Dobson, A. P., Ostfeld, R. S., & Samuel, M. D. (2002). Climate warming and disease risks for terrestrial and marine biota. Science. https://doi.org/10.1126/science.1063699 Heil, M. (2016). Host manipulation by parasites: Cases, patterns, and remaining doubts. Frontiers in Ecology and Evolution, 4, 80. https://doi.org/10.3389/fevo.2016.00080 Hellgren, O., Pérez-Tris, J., & Bensch, S. (2009). A jack-of-all-trades and still a master of some: Prevalence and host range in avian malaria and related blood parasites. Ecology, 90(10), 2840–2849. https://doi.org/10.1890/08-1059.1 Hellgren, O., Waldenström, J., & Bensch, S. (2004). A new PCR assay for simultaneus studies of Leucocytozoon, Plasmodium and Haemoproteus from avian blood. Journal of Parasitology, 90(4), 797–802. https://doi.org/10.1645/GE-184R1 Holdich, D. M., & Reeve, I. D. (1991). Distribution of freshwater crayfish in the British Isles, with particular reference to crayfish plague, alien introductions and water quality. Aquatic Conservation: Marine and Freshwater Ecosystems, 1(2), 139–158. https://doi.org/10.1002/aqc.3270010204 Jia, T., Huang, X., Valkiūnas, G., Yang, M., Zheng, C., Pu, T., … Zhang, C. (2018). Malaria parasites and related haemosporidians cause mortality in cranes: A study on the parasites diversity, prevalence and distribution in Beijing Zoo. Malaria Journal, 17(1), 234. https://doi.org/10.1186/s12936-018-2385-3

40 Klein, S. L. (2004, June 1). Hormonal and immunological mechanisms mediating sex differences in parasite infection. Parasite Immunology. https://doi.org/10.1111/j.0141-9838.2004.00710.x Knowles, S. C. L., Palinauskas, V., & Sheldon, B. C. (2010). Chronic malaria infections increase family inequalities and reduce parental fitness: Experimental evidence from a wild bird population. Journal of Evolutionary Biology, 23(3), 557–569. https://doi.org/10.1111/j.1420-9101.2009.01920.x Kulma, K., Low, M., Bensch, S., & Qvarnström, A. (2013). Malaria infections reinforce competitive asymmetry between two Ficedula flycatchers in a recent contact zone. Molecular Ecology, 22, 4591–4601. https://doi.org/10.1111/mec.12409 LaPointe, D. A., Atkinson, C. T., & Samuel, M. D. (2012). Ecology and conservation biology of avian malaria. Annals of the New York Academy of Sciences, 1249(1), 211–226. https://doi.org/10.1111/j.1749-6632.2011.06431.x Le Galliard, J.-F., Fitze, P. S., Ferrière, R., & Clobert, J. (2005). From the cover: Sex ratio bias, male aggression, and population collapse in lizards. Proceedings of the National Academy of Sciences, 102(50), 18231–18236. https://doi.org/10.1073/pnas.0505172102 Logan, J. G., Birkett, M. A., Clark, S. J., Powers, S., Seal, N. J., Wadhams, L. J., … Pickett, J. A. (2008). Identification of human-derived volatile chemicals that interfere with attraction of Aedes aegypti mosquitoes. Journal of Chemical Ecology, 34(3), 308–322. https://doi.org/10.1007/s10886-008-9436-0 Low, M., Arlt, D., Eggers, S., & Pärt, T. (2010). Habitat-specific differences in adult survival rates and its links to parental workload and on-nest predation. Journal of Animal Ecology, 79(1), 214–224. https://doi.org/10.1111/j.1365- 2656.2009.01595.x Lundberg, A., & Alatalo, R. V. (1992). The Pied Flycatcher. London: T & AD Poyser. Maklakov, A. A., & Lummaa, V. (2013). Evolution of sex differences in lifespan and aging: Causes and constraints. BioEssays. https://doi.org/10.1002/bies.201300021 Martínez-De La Puente, J., Merino, S., Tomás, G., Moreno, J., Morales, J., Lobato, E., … Sarto I Monteys, V. (2009). Factors affecting Culicoides species composition and abundance in avian nests. Parasitology, 136(9), 1033–1041. https://doi.org/10.1017/S0031182009006374 Marzal, A., Balbontín, J., Reviriego, M., García-Longoria, L., Relinque, C., Hermosell, I. G., … Møller, A. P. (2016). A longitudinal study of age-related changes in Haemoproteus infection in a passerine bird. Oikos, 125(8), 1092– 1099. https://doi.org/10.1111/oik.02778 Marzal, A., Ricklefs, R. E., Valkiūnas, G., Albayrak, T., Arriero, E., Bonneaud, C., … Bensch, S. (2011). Diversity, loss, and gain of malaria parasites in a globally invasive bird. PLoS ONE, 6(7), e21905. https://doi.org/10.1371/journal.pone.0021905 McCurdy, D. G., Shutler, D., Mullie, A., & Forbes, M. R. (1998). Sex-biased parasitism of avian hosts: Relations to blood parasite taxon and mating system. Oikos, 82(2), 303. https://doi.org/10.2307/3546970 Medeiros, M. C. I., Ellis, V. A., & Ricklefs, R. E. (2014). Specialized avian Haemosporida trade reduced host breadth for increased prevalence. Journal of Evolutionary Biology, 27(11), 2520–2528. https://doi.org/10.1111/jeb.12514 Millar, J. G., & Sims, J. J. (1998). Preparation, cleanup and preliminary fractionation of extracts. In Methods in chemical ecology (pp. 1–37). Boston: Kluwer Academic Publishers.

41 Montgomerie, R. D., & Weatherhead, P. J. (1988). Risks and rewards of nest defence by parent birds. The Quarterly Review of Biology, 63(2), 167–187. https://doi.org/10.1086/415838 Nordling, D., Andersson, M., Zohari, S., & Gustafsson, L. (1998). Reproductive effort reduces specific immune response and parasite resistance. Proceedings of the Royal Society B: Biological Sciences, 265(1403), 1291–1298. https://doi.org/10.1098/rspb.1998.0432 Park, T. (1948). Interspecies competition in populations of Trilobium confusum Duval and Trilobium castaneum Herbst. Ecological Monographs, 18(2), 265– 307. https://doi.org/10.2307/1948641 Parmesan, C. (2006). Ecological and evolutionary responses to recent climate change. Annual Review of Ecology, Evolution, and Systematics, 37(1), 637–669. https://doi.org/10.1146/annurev.ecolsys.37.091305.110100 Parmesan, C., & Yohe, G. (2003). A globally coherent fingerprint of climate change impacts across natural systems. Nature, 421(6918), 37–42. https://doi.org/10.1038/nature01286 Post, P., & Götmark, F. (2006). Predation by sparrowhawks Accipiter nisus on male and female pied flycatchers Ficedula hypoleuca in relation to their breeding behaviour and foraging. Journal of Avian Biology, 37(2), 158–168. https://doi.org/10.1111/j.0908-8857.2006.03338.x Poulin, R. (2011). Evolutionary ecology of parasites. Princeton University Press. Qvarnström, A., Wiley, C., Svedin, N., & Vallin, N. (2009). Life-history divergence facilitates regional coexistence of competing Ficedula flycatchers. Ecology, 90(7), 1948–1957. https://doi.org/10.1890/08-0494.1 Råberg, L., Sim, D., & Read, A. F. (2007). Disentangling genetic variation for resistance and tolerance to infectious diseases in animals. Science, 318(5851), 812–814. https://doi.org/10.1126/science.1148526 Reullier, J., Pérez-Tris, J., Bensch, S., & Secondi, J. (2006). Diversity, distribution and exchange of blood parasites meeting at an avian moving contact zone. Molecular Ecology, 15(3), 753–763. https://doi.org/10.1111/j.1365- 294X.2005.02826.x Roy, B., & Kirchner, J. (2000). Evolutionary dynamics of pathogen resistance and tolerance. Evolution, 54(1), 51–63. https://doi.org/10.1554/0014-3820(2000)054 Rushton, S. P., Lurz, P. W. W., Gurnell, J., Nettleton, P., Bruemmer, C., Shirley, M. D. F., & Sainsbury, A. W. (2006). Disease threats posed by alien species: The role of a poxvirus in the decline of the native red squirrel in Britain. Epidemiology and Infection, 134(3), 521–533. https://doi.org/10.1017/S0950268805005303 Rybinski, J., Sirkiä, P. M., McFarlane, S. E., Vallin, N., Wheatcroft, D., Ålund, M., & Qvarnström, A. (2016). Competition-driven build-up of habitat isolation and selection favoring modified dispersal patterns in a young avian hybrid zone. Evolution, 70(10), 2226–2238. https://doi.org/10.1111/evo.13019 Sætre, G. P., Borge, T., Lindell, J., Moum, T., Primmer, C. R., Sheldon, B. C., … Ellegren, H. (2001). Speciation, introgressive hybridization and nonlinear rate of molecular evolution in flycatchers. Molecular Ecology, 10(3), 737–749. https://doi.org/10.1046/j.1365-294X.2001.01208.x Sætre, G. P., Kral, M., Bures, S., & Ims, R. A. (1999). Dynamics of a clinal hybrid zone and a comparison with island hybrid zones of flycatchers (Ficedula hypoleuca and F-albicollis). Journal of Zoology, 247(1), 53–64. https://doi.org/10.1111/j.1469-7998.1999.tb00192.x

42 Salewski, V., Bairlein, F., & Leisler, B. (2002). Different wintering strategies of two Palearctic migrants in West Africa - a consequence of foraging strategies? Ibis, 144(1), 85–93. https://doi.org/10.1046/j.0019-1019.2001.00007.x Schall, J. J. (1992). Parasite-mediated competition in Anolis lizards. Oecologia, 92(1), 58–64. https://doi.org/10.1007/BF00317262 Schmid-Hempel, P. (2010). Evolutionary parasitology. The integrated study of infections, immunology, ecology, and genetics. Oxford University Press, New York Scordato, E. S. C., & Kardish, M. R. (2014). Prevalence and beta diversity in avian malaria communities: Host species is a better predictor than geography. Journal of Animal Ecology, 83(6), 1387–1397. https://doi.org/10.1111/1365-2656.12246 Smith, L. M., & Burgoyne, L. A. (2004). Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper. BMC Ecology, 4(1), 4. https://doi.org/10.1186/1472-6785-4-4 Sol, D., Jovani, R., & Torres, J. (2003). Parasite mediated mortality and host immune response explain age-related differences in blood parasitism in birds. Oecologia, 135(4), 542–547. https://doi.org/10.1007/s00442-003-1223-6 Sonerud, G. A. (1985). Risk of nest predation in three species of hole nesting owls: Influence on choice of nesting habitat and incubation behaviour. Ornis Scandinavica, 16(4), 261–269. https://doi.org/10.2307/3676689 Stevenson, I. R., & Bryant, D. M. (2000). Avian phenology: Climate change and constraints on breeding. Nature, 406(6794), 366–367. https://doi.org/10.1038/35019151 Stjernman, M., Råberg, L., & Nilsson, J.-A. (2004). Survival costs of reproduction in the blue tit (Parus caeruleus): a role for blood parasites? Proceedings. Biological Sciences / The Royal Society, 271(1555), 2387–2394. https://doi.org/10.1098/rspb.2004.2883 Suárez, J. (2018). The importance of symbiosis in philosophy of biology: an analysis of the current debate on biological individuality and its historical roots. Symbiosis. https://doi.org/10.1007/s13199-018-0556-1 Svedin, N., Wiley, C., Veen, T., Gustafsson, L., & Qvarnström, A. (2008). Natural and sexual selection against hybrid flycatchers. Proceedings of the Royal Society B: Biological Sciences, 275(1635), 735–744. https://doi.org/10.1098/rspb.2007.0967 Thomas, F., Poulin, R., & Brodeur, J. (2010). Host manipulation by parasites: A multidimensional phenomenon. Oikos, 119(8), 1217–1223. https://doi.org/10.1111/j.1600-0706.2009.18077.x Tower, J. (2006). Sex-specific regulation of aging and apoptosis. Mechanisms of Ageing and Development, 127(9), 705–718. https://doi.org/10.1016/j.mad.2006.05.001 Trivers, R. (1972). Parental investment and sexual selection. Cambridge MA: Biological Laboratories, Harvard University. Trivers, R. (1985). Social Evolution. Benjamin Cummings, Boston, MA. Valkiūnas, G. (2005). Avian Malaria Parasites and other Haemosporidia. Boca Raton, FL: CRC Press. https://doi.org/10.1201/9780203643792 Valkiūnas, G., Liutkevičius, G., & Iezhova, T. A. (2002). Complete development of three species of Haemoproteus (Haemosporidia, Haemoroteidae) in the biting midge Culicoides impunctatus (Diptera, Ceratopogonidae). Journal of Parasitology, 88(5), 864–868. https://doi.org/10.1645/0022-3395(2002)088

43 Vallin, N., Rice, A. M., Arntsen, H., Kulma, K., & Qvarnström, A. (2012). Combined effects of interspecific competition and hybridization impede local coexistence of Ficedula flycatchers. Evolutionary Ecology, 26(4), 927–942. https://doi.org/10.1007/s10682-011-9536-0 van Riper, C., van Riper, S. G., Goff, M. L., & Laird, M. (2006). The epizootiology and ecological significance of malaria in Hawaiian land birds. Ecological Monographs, 56(4), 327–344. https://doi.org/10.2307/1942550 Waldenström, J., Bensch, S., Hasselquist, D., & Östman, Ö. (2004). A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. The Journal of Parasitology, 90(1), 191–194. https://doi.org/10.1645/GE-3221RN Walther, G.-R., Post, E., Convey, P., Menzel, A., Parmesan, C., Beebee, T. J. C., … Bairlein, F. (2002). Ecological responses to recent climate change. Nature, 416(6879), 389–395. https://doi.org/10.1038/416389a Wesołowski, T. (2002). Anti-predator adaptations in nesting mrsh tits Parus palustris: The role of nest-site security. Ibis, 144(4), 593–601. https://doi.org/10.1046/j.1474-919X.2002.00087.x Wiebe, K. L. (2004). Innate and learned components of defence by flickers against a novel nest competitor, the European starling. Ethology, 110(10), 779–791. https://doi.org/10.1111/j.1439-0310.2004.01016.x Williams, G. C. (1957). Pleiotrophy, natural selection, and the evolution of senescence. Evolution, 11(4), 398–411. https://doi.org/10.2307/2406060 Woodworth, B. L., Atkinson, C. T., LaPointe, D. A., Hart, P. J., Spiegel, C. S., Tweed, E. J., … Duffy, D. (2005). Host population persistence in the face of introduced vector-borne diseases: Hawaii amakihi and avian malaria. Proceedings of the National Academy of Sciences, 102(5), 1531–1536. https://doi.org/10.1073/pnas.0409454102 Zuk, M., & McKean, K. A. (1996). Sex differences in parasite infections: Patterns and processes. International Journal for Parasitology. https://doi.org/10.1016/S0020-7519(96)00086-0

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