Selection on Morphological Traits and Fluctuating Asymmetry by a 9 Fungal Parasite in the Yellow Dung Fly

bioRxiv preprint doi: https://doi.org/10.1101/136325; this version posted January 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

1 2 3 4 5 6 7 Blanckenhorn WU. 2017. 8 Selection on morphological traits and fluctuating asymmetry by a 9 fungal parasite in the yellow dung fly. 10 bioRxiv 136325, http://dx.doi.org/10.1101/136325 11 12 A Preprint reviewed and recommended by Peer Community 13 Evolutionary Biology: 14 http://dx.doi.org/10.24072/pci.evolbiol.100027 15

16 17

Selection on morphological traits and fluctuating asymmetry by a fungal parasite in the yellow dung fly

WOLF U. BLANCKENHORN

Department of Evolutionary Biology and Environmental Studies, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; Fax: +41 44 635.4780; E-mail: [email protected]

Abstract A Preprint reviewed and recommended by Peer Community Evolutionary Biology: http://dx.doi.org/10.24072/pci.evolbiol.100027

Evidence for selective disadvantages of large body size remains scarce in general. Previous phenomenological studies of the yellow dung fly Scathophaga stercoraria have demonstrated strong positive sexual and fecundity selection on male and female size. Nevertheless, the body size of flies from a Swiss study population has declined by almost 10% from 1993 to 2009. Given substantial heritability of body size, this negative evolutionary response of an evidently positively selected trait suggests important selective factors being missed (e.g. size-selective predation or parasitism). A periodic epidemic outbreak of the fungus Entomophthora scatophagae allowed assessment of selection exerted by this parasite fatal to adult flies. Fungal infection varied over the season from ca. 50% in the cooler and more humid spring and autumn to almost 0% in summer. The probability of dying from fungal infection increased with adult body size. All infected females died before laying eggs, so there was no fungus impact on female fecundity beyond its impact on mortality. Large males showed the typical mating advantage in the field, but this pattern of positive sexual selection was nullified by fungal infection. Mean fluctuating asymmetry of paired appendages (legs, wings) did not affect the viability, fecundity or mating success of yellow dung flies in the field. This study demonstrates rare parasite-mediated disadvantages of large adult body size in the field. Reduced ability to combat parasites such as Entomophthora may be an immunity cost of large size in dung flies, although the hypothesized trade-off between fluctuating asymmetry, a presumed indicator of developmental instability and environmental stress, and immunocompetence was not found here.

Keywords: body size, developmental stability, Entomophthora, fecundity selection, fluctuating asymmetry, fungal parasite, insect immunity, Scathophaga stercoraria, sexual selection, trade- off, viability selection.

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Introduction stercoraria (Diptera: Scathophagidae) is a Systematic quantification of selection has classic model species for studies of natural, become one of the hallmarks of modern particularly sexual selection (Parker, 1979; biological research so as to acquire a Borgia, 1982; Sigurjónsdóttir & Snorrason, thorough understanding of the process of 1995) and a typical case in point. Field natural selection and its evolutionary studies have established temporally consequences. Standardized measures of variable but on average very strong mating selection (Arnold & Wade, 1984a,b; Lande advantages of large males, as well as & Arnold, 1983; Brodie et al., 1995) have fecundity advantages of large females (Jann been available for some time and applied to et al., 2000; Blanckenhorn et al., 2003; cf. many species and situations to foster Honek, 1993). Strong sexual selection on multiple comparative (meta-)analyses, male body size likely is the main driver of which greatly enhanced our understanding the untypical male-biased sexual size of the action of natural selection in the wild dimorphism in this species (Fairbairn, (e.g. Endler, 1986; Kingsolver et al., 2001; 1997; Kraushaar & Blanckenhorn, 2002; Kingsolver & Pfennig, 2004; Cox & Blanckenhorn, 2007, 2009). Nevertheless, Calsbeek, 2009). Correlative the body size of flies from our field phenomenological investigations of population in Switzerland has declined by selection also are the field method of choice almost 10% over a 15-year period from to understand the evolution and population 1993 – 2009 (Blanckenhorn, 2015). Given biology of single species or populations in generally substantial heritability of body an integrative way, and to test hypotheses size also in this species (Mousseau & Roff, about the evolution of particular traits and 1987; Blanckenhorn, 2000), this negative patterns (e.g. sexual size dimorphism: evolutionary response of a trait that is Blanckenhorn, 2007). measurably strongly positively selected A common pattern is that in many suggests that we are missing important species of animals fecundity selection selective factors or episodes shaping the favors large females and sexual selection body size of yellow dung flies (Merilä et al., large males (Roff, 1992; Andersson, 1994; 2001; Blanckenhorn, 2015; Gotanda et al., Kingsolver & Pfennig, 2004). Large body 2015). In general, and also for the yellow size also confers a number viability benefits dung fly, evidence for selective (e.g. a longer life), so the selective forces disadvantages of large body size remains favoring small body size, presumably scarce (Blanckenhorn, 2000, 2007). primarily related to juvenile viability costs One aspect not well studied in yellow of becoming large and/or costs of dung flies is size-dependent survival in maintaining a large adult size, are often nature. This is generally the case for small- cryptic (Blanckenhorn, 2000). The bodied invertebrates, for which longitudinal widespread yellow dung fly Scathophaga field studies are essentially impossible bioRxiv preprint doi: https://doi.org/10.1101/136325; this version posted January 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Wolf U. Blanckenhorn Selection by a fungal parasite in dung flies

because individuals cannot be easily Nielsen & Hayek, 2006). This study took marked and followed in nature, as is the advantage of a fungus epidemic at our field case for larger vertebrates (Merilä & population near Zürich, Switzerland, in Hendry, 2014, Schilthuizen & Kellermann, 2002. 2014; Stoks et al., 2014; Blanckenhorn, 2015). At the same time, laboratory I here assess viability, fecundity and longevity estimates (e.g. Blanckenhorn, sexual selection on morphology and 1997; Reim et al., 2006; Blanckenhorn et fluctuating asymmetry. Morphological al., 2007) generally do not well reflect field traits reflecting body size are often assessed mortality. Multiple field estimates of larval in selection studies (Kingsolver et al., 2001; survivorship at various conditions exist Kingsolver & Pfennig, 2004). Body size is suggesting some counter-selection against one of the most important quantitative traits large body size via the necessarily longer of an organism, as it strongly affects most development time (summarized in physiological and fitness traits (Calder, Blanckenhorn, 2007). However, sex- and 1984; Schmidt-Nielsen, 1984; Roff, 1992) size-specific adult survivorship in the field and exhibits several prominent evolutionary was so far estimated only indirectly by patterns in many organisms (Rensch, 1950; Burkhard et al. (2002) using age-grading by Fairbairn 1997; Blanckenhorn, 2000; wing injuries. The present study assesses Blanckenhorn & Demont, 2004; Kingsolver natural selection on morphological traits by & Pfennig, 2004). Depending on the taxon, the fatal fungal parasite Entomophthora diverse traits are typically used as spp. surrogates of body size, which are usually The parasitic fungus Entomophthora highly correlated (i.e. integrated) within scatophagae regularly infects yellow dung individuals due to pleiotropy, epistasis or fly adults in Europe and North America gene linkage. Nevertheless, for functional (Hammer, 1941; Steinkraus & Kramer, reasons selection on various body parts may 1988; Maitland, 1994; Steenberg et al., differ (e.g. Preziosi & Fairbairn, 2000; 2001). Primarily at humid conditions Fairbairn, 2005), producing responses in infections can be epidemic (pers. obs.), in correlated traits and thus generally which case infected dead flies can be found prompting a multivariate approach (Lande prominently exposed near cow pastures on & Arnold, 1983). I focused on paired flowers, long grass or fences in a appendages (legs, wings), so I could also characteristic posture presumably assess fluctuating asymmetry (FA; Palmer effectively disseminating fungal spores & Strobeck, 1986). Small and random and/or attracting other flies (Maitland, deviations from the a priori perfect 1994; Møller, 1993). Spore transmission symmetry in bilaterally symmetric likely also occurs via physical contact, e.g. organisms, i.e. FA, are presumed to reflect during copulation (Møller, 1993). The heritable developmental instability, such fungus is fierce and effective at infecting that individuals with good genes and/or and killing adult flies within hours or days living in good conditions can produce more and can be manipulated, such that related, symmetric bodies in the face of often species-specific such fungi are being environmental stress, ultimately employed for biological control of some augmenting their fitness. Symmetric insect pests (e.g. Steenberg et al., 2001; individuals consequently should have

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greater survival prospects (viability season (September – November), while selection), and should be more successful at during the hot midsummer (July and acquiring mates (sexual selection: Møller & August) the flies largely disappear from the Swaddle, 1997). However, especially the pastures due to their heat sensitivity latter notion, and the evidence, remain (Blanckenhorn, 2009). controversial (Møller & Thornhill, 1997; Adult S. stercoraria are sit-and-wait Palmer, 2000; Polak, 2003; Klingenberg, predators of small flying insects, from 2003; Van Dongen, 2006; Knierim et al., which they extract protein to produce sperm 2007). In yellow dung flies, Liggett et al. and eggs (Foster, 1967). Females spend (1993) and Swaddle (1997) found a most of their time foraging for nectar and negative relationship between FA and prey in the vegetation surrounding pastures. mating and foraging success, respectively, About once a week they visit fresh cattle (or while Floate & Coughlin (2010) found no other) dung to deposit a clutch of eggs. evidence for FA being a useful biomarker Larvae feed on and develop in the dung. of environmental stress exerted by toxic Multiple males typically wait at the dung livestock medications (ivermectin). Our pat to mate with incoming females. previous studies of this species found that Copulation usually takes place in the FA is not heritable (Blanckenhorn & surrounding grass or on the dung pat; during Hosken, 2003), that it does not increase the ensuing oviposition the male guards the with inbreeding (or homozygosity: Hosken female against other competitors (Parker, et al., 2000), and that FA does not affect 1970). Competition among males for male mating success in the field, while females is very strong as the operational sex nevertheless being negatively related to ratio is highly male biased (Jann et al., energy reserves (Blanckenhorn et al., 2000). Larvae face unpredictable spatio- 2003). Of key relevance here is the temporal variation in local temperatures, postulated link between FA and dung (i.e. food) quality and quantity, intra- immunocompetence, predicting that more and inter-specific competition, and dung symmetric, but likely also larger individuals drying, all factors that ultimately largely are better at fending off internal parasites determine their phenotypic adult body size. such as Entomophthora (see e.g. Rantala et Towards the end of the season the flies have al., 2000, 2004, 2007 and Yourth et al., to reach the overwintering pupal stage 2002, for various insect species), which was before the first winter frost (Blanckenhorn, tested here. 2009).

Fly sampling Material and Methods I sampled our population in Fehraltorf near Zurich (N47º52’, E8º44’) roughly once a Study species month between April and November 2002 Yellow dung flies occur throughout the (8 seasonal samples; total of N = 541 flies). northern hemisphere and are particularly Each time I sampled one randomly selected common around cow pastures in central but otherwise representative fresh dung pat, Europe. The species prefers cooler climates. collecting all single and paired flies on and In lowland central Europe, each year has a ca. 20 cm around the pat to bring them alive spring (March – June) and an autumn to the laboratory. As virtually no unpaired

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females occur at a pat in the field because Statistical analysis competition for mates is so intense, the For each monthly sample, and overall, I number of pairs corresponds to the number calculated standardized viability selection of females present, and the proportion of differentials (= gradients) for both sexes paired males corresponds to the operational (binary variable: dead/alive = sex ratio (females / males). infected/uninfected), sexual selection differentials for males (binary: Laboratory procedures mated/unmated), and fecundity selection Although dead flies infected with the gradients for females based on their clutch fungus were occasionally found on and size, using standard methods (Lande & around the pasture, these were too rare and Arnold, 1983; Arnold & Wade, 1984a,b; haphazard to be sampled systematically. Brodie et al., 1995). It turns out that almost Instead, all collected flies were kept alive in all females that developed the fungus and the laboratory in single 100 ml bottles with eventually died in the laboratory did not lay sugar and water for up to two weeks. any eggs, so fecundity selection coefficients Infected flies would develop the fungus only refer to healthy (uninfected) females. within few days and eventually die; non- Selection coefficients were calculated for infected, recovered or resistant flies would each trait (4 morphological and 4 not (acknowledging some random asymmetry traits) separately for females background mortality). Females received and males, and additionally for the first dung to oviposit one clutch of eggs, which principal component (PC) of all (mean) was counted, which yields a good estimate appendages signifying a compound index of of body size dependent clutch size (Honek, body size, as such compound indices are 1993; ca. 30 – 90 eggs: Jann et al., 2000; often preferred by some researchers as Blanckenhorn, 2007). proxies of overall body size even though In the end, work study students they are redundant in this context. measured left and right wing length as well Because the sizes of all appendages were as fore, mid and hind tibia length of each highly positively correlated, and because fly. Mean values for these paired traits were FA and size are mathematically related (see subsequently calculated, as well as signed formulae above), we calculated only FA as (L – R), unsigned FA as (|L – R|) univariate linear (buni) and corresponding (both in mm) and unsigned, size-corrected non-linear (guni) selection coefficients. To FA as (|L – R|) / mean(L, R) in %, as do so, for each seasonal sample we suggested by Palmer & Strobeck (1986). produced standardized z-scores for trait x by Paired traits were measured twice blindly subtracting the sample mean from each by the same person to estimate value and dividing by the standard measurement error relative to fluctuating deviation: . Relative asymmetry (Palmer & Strobeck, 1986), and to calculate the repeatability of all trait survival or male pairing success was measurements (Becker, 1992). All computed as absolute survival or pairing measurements were taken with a binocular success (1 or 0) divided by the sample microscope at 16x magnification. proportion of survived flies or mated males, respectively (Brodie & Janzen, 1996), and relative clutch size as absolute clutch size

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divided by mean clutch size. We used the beginning (spring) and the end of the season univariate model of relative fitness on (autumn), whereas they were rare during the standardized body size to hotter summer (nearly 0%); females were estimate the linear selection intensities, and more affected than males (significant sex by the corresponding quadratic model season interaction: �2 = 29.80; P < 0.001; to estimate Fig. 1). The probability of dying by fungal 2 univariate non-linear selection intensities, infection was unaffected by mean FA (� = . These linear coefficients (gradients) 1.99; P = 0.159) but increased with fly body 2 reflect the combined effects of direct and size (� = 12.56; P < 0.001; Table 1), an indirect selection on body size (Endler, effect that however varied among seasonal 2 1986). The difference of the regression samples (� = 12.55; P < 0.001) but not 2 coefficients from a slope of zero (the null between the sexes (� = 0.27; P = 0.602). hypothesis of no selection) was tested. For Fecundity selection (based on clutch estimation of the coefficients least-squares size) on female body size was significantly regression was applied, but for tests of positive, as is typical in this species (Jann et significance logistic regression was used in al., 2000; Kraushaar & Blanckenhorn, case our measures of success were binary 2002). The intensity of fecundity selection, (viability and mating success: Brodie et al., i.e. the slope relating relative clutch size to 1995; cf. Janzen & Stern, 1998). A full standardized body size (PC), varied model with sex and season (including significantly but unsystematically over the

interactions) was performed to test for season (Table 1; interaction test: F6,109 = variation in selection across these main 3.50, P = 0.03). These estimates refer only factors. to uninfected flies because all females infected with the fungus died before laying eggs, and therefore do not estimate fecundity selection exerted by the fungus beyond the parasite’s effect on adult mortality.

Figure 1: Proportion of male (filled squares) and female (open circles) flies infected by the fungus Entomophthora over the season 2002.

Results Fungus prevalence varied over the season and between the sexes. Infections (as high as 50%) were most common during the cooler and more humid periods at the

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�2 = 7.61; P = 0.006; fungus by size interaction: �2 = 3.75; P = 0.053; Fig. 3).

Figure 3: Body size (here exemplified by hind tibia length) of unpaired (filled squares) and paired males (open squares) when they were infected by the fungus or not (all seasonal samples combined).

Table 1 gives directional (linear) Figure 2: Body size (top; here exemplified by wing selection coefficients, �, for the overall length) and mean percentage of fluctuating asymmetry (FA; bottom) of all traits for unpaired sample. Nonlinear (quadratic) selection (filled squares) and paired males (open squares) over coefficients, �, were mostly low and not the season. significant and are therefore not presented

As usual in yellow dung flies, larger in Table 1. The only exception was female males had a mating advantage (Jann et al., fecundity selection on body size, for which 2000; Kraushaar & Blanckenhorn, 2002; � = 0.056 ± 0.025 was significantly Blanckenhorn et al., 2003; main effect of positive, signifying accelerating selection body size (PC): �2 = 14.23; P < 0.001), (which has been reported before: while mean FA of all paired appendages did Blanckenhorn, 2007, 2009). Leg and wing lengths were expectedly highly correlated not affect male mating success (�2 = 1.17; P in both sexes (range of bivariate = 0.279; Table 1; Fig. 2). Except for one correlations: r = 0.887 to 0.972), whereas seasonal sample on 29 May, the large male FA of the legs and wings were largely advantage was consistent throughout the uncorrelated (range: r = -0.024 to +0.215). season such that sexual selection intensity Measurement of all paired traits was did not significantly vary across the season generally repeatable using our methods (R (body size by season interaction: �2 = 0.11; = 0.83 – 0.97), which was also true for P = 0.920; Table 1; Fig. 2). Interestingly, asymmetry (R = 0.53 – 0.61), so that FA this pattern of positive sexual selection was could be discerned from measurement error nullified by fungal infection in those 28 (of (all side by individual interactions P <0.01), a total of 47) infected males (of a total of fulfilling the criteria of proper FA 370 males, of which 148 had a mate) found assessment (Palmer & Strobeck, 1986; mating in the field that later succumbed to Knierim et al., 2007). the fungus (main effect of fungal infection:

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Discussion consistent negative viability selection on At our Swiss study population, the female and male adult body size in S. entomophagous fungal parasite stercoraria in those parts of the year, when Entomophthora scatophagae, which has dung flies are most common (Table 1). been described as a specific parasite of adult Fungal infection further nullifies the usual yellow dung flies at several sites in Europe large male mating advantage in sexual and North America (Hammer, 1941; selection (Borgia, 1982; Jann et al. 2000; Steinkraus & Kramer, 1988; Maitland, Blanckenhorn et al., 2003; Fig. 3), but does 1994; Steenberg et al., 2001), shows high not affect female fecundity beyond its and generally fatal infection rates of up to impact on mortality. This represents the 50% during the cooler and more humid first evidence demonstrating viability periods of the year. I here documented that disadvantages of large yellow dung flies this fungus exerts relatively strong and mediated by a parasite, which is generally

Table 1: Overall intensities (� ± 95% CI) of female and male adult viability selection (Nf = 171 & Nm = 370) exerted by the fungus Entomophthora, female fecundity selection (clutch size; Nf = 126), and male sexual selection (pairing success; Nm = 370) for one Swiss population of yellow dung flies (Scathophaga stercoraria) over the season 2002. Significant coefficients are in bold (P < 0.001).

rare in animals and particularly reduced parasite resistance of large flies invertebrates (Blanckenhorn, 2000; signifies a trade-off between body size and Kingsolver & Pfennig, 2004; Gotanda et al., immunity (Rantala & Roff, 2005; 2015). These results complement previous Schwarzenbach &Ward, 2006; Cotter et al., evidence of viability disadvantages of large 2008). Based on age grading by wing flies at the juvenile stage, and lend further injuries, Burkhard et al. (2002) found that credence to the notion that the male-biased adult age (i.e. longevity) of yellow dung sexual size dimorphism of yellow dung flies flies in the field tends to be positively is indeed roughly at evolutionary related to body size. Energy reserves also equilibrium (Blanckenhorn, 2007). scale positively with body size (Reim et al., This study is merely phenomenological, 2006; Blanckenhorn et al., 2007) and meaning that mechanisms were not positively influence mating success assessed. Nonetheless, I speculate that the (Blanckenhorn et al., 2003). Adult

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longevity under most environmental selection here turned out to be steeper in circumstances, including complete males (Table 1). Yellow dung fly females starvation, can therefore generally be indeed produce higher heritable levels than expected to increase with body size on males of phenoloxidase (PO; physiological grounds. One possible Schwarzenbach et al., 2005), one of the mechanism selecting against large body central mediators of insect immunity size is size-selective predation and/or (Schmid-Hempel, 2005; Rolff & Reynolds, parasitism (Blanckenhorn, 2000). However, 2009; González-Santoyo & beyond expectations of a rough positive CórdobaAguilar, 2012), and higher PO correlation between predator and prey size levels decrease adult longevity in this (Brose et al., 2006; Vucic-Pestic et al., species, demonstrating a trade-off 2010), evidence for systematic size- (Schwarzenbach & Ward, 2006). However, selectivity of predators is generally weak at higher PO levels did not lead to greater best, also for yellow dung flies resistance against mites or another fungus (Blanckenhorn, 2000; Blanckenhorn et al., (Schwarzenbach & Ward, 2007), and PO is unpublished data). Size-selective parasitism also unrelated to body size in S. stercoraria has been reported in some parasitoids (Schwarzenbach et al., 2005). Overall, (McGregor & Roitberg, 2000), but therefore, the evidence in favor of immunity otherwise few data exist (Zuk & Kolluru, mediating the higher mortality of large- 1998; Blanckenhorn, 2000). Rather than bodied dung flies here remains limited. invoking selective attraction of the parasite In contrast to body size, fluctuating by larger hosts, I suspect that the ability to asymmetry (FA) of legs and wings combat the parasite is compromised in influenced none of the fitness components larger flies due to their generally greater investigated here (contrary to Liggett et al., absolute energy demands in a stressful 1993, but confirming Blanckenhorn et al’s, environment (trade-off hypothesis: Rantala 2003, earlier sexual selection study). I had & Roff, 2005; Reim et al., 2006; expected, based on evidence in other Schwarzenbach & Ward, 2006, 2007; animals (Rantala et al., 2000, 2004), that Cotter et al., 2008). Females were infected low FA would be a signal of greater more by the parasite here (Fig. 1), probably immunocompetence augmenting resistance related to their generally greater against parasites, but this was not found. It reproductive burden (i.e. the cost of is not unlikely that FA is a bad indicator of producing expensive eggs rather than cheap developmental stability in general, as sperm; cf. Nunn et al., 2009; but see e.g. various reviews have revealed no clear Rantala et al., 2007, for opposite results). verdict based on the available evidence on Nevertheless, the standard sex differences this question, so the entire concept remains in reproductive (energetic) costs should be controversial (Møller & Thornhill, 1997; somewhat offset by the male-biased sexual Møller & Swaddle, 1997; Palmer, 2000; size dimorphism of yellow dung flies, various articles in Polak, 2003; Van which implies relatively greater costs when Dongen, 2006; Knierim et al., 2007). In producing and maintaining the larger and yellow dung flies, beyond Liggett et al. condition-dependent male body (1993) the evidence for a role of FA in (Blanckenhorn, 2000, 2007), and might sexual selection is close to nil explain why size-dependent viability (Blanckenhorn et al., 2003; Blanckenhorn

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& Hosken, 2003; this study). What remains is that FA reliably indicates at least hot temperature stress in this species (Hosken et al., 2000; Blanckenhorn & Hosken, further unpublished data), even though Floate & Coughlin (2010) concluded that FA is no good biomarker of toxic chemical residues in cattle dung. As the survival of flies infected with the fierce entomophagous fungus Entomophthora scatophagae here was related to the size (discussed above) but not the fluctuating asymmetry of wings and legs, I conclude that FA is no good indicator of immunocompetence in yellow dung flies either (Rantala et al., 2000, 2004, 2007; Rantala & Roff, 2005; Yourth et al., 2002; Schwarzenbach &Ward, 2006; Cotter et al., 2008).

Acknowledgements I thank the University of Zurich and the Swiss National Fund for financial support over the years, and Ana, Lukas, Nils and Patricia for doing the measurements.

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References Borgia, G. 1982. Experimental changes in resource structure and male density: size- Andersson. M. 1994. Sexual Selection. Princeton related differences in mating success among University Press, Princeton, NJ. male Scathophaga stercoraria. Evolution 36: Arnold, S. J., & Wade, M.J. 1984a. On the 307-315. measurement of natural and sexual selection: Brodie III., E.D. & Janzen, F.J. 1996. On the applications. Evolution 38: 720-734. assignment of fitness values in statistical Arnold, S. J., & Wade, M.J. 1984b. On the analyses of selection. Evolution 50: 437-442. measurement of natural and sexual selection: Brodie III., E.D., Moore, A.J. & Janzen, F.J. 1995. theory. Evolution 38: 709-719. Visualizing and quantifying natural selection. Becker, W.A. 1992. Manual of Quantitative Trends Ecol. Evol. 10: 313-318. Genetics. 5th edition. Academic Enterprises, Brose U, et al. 2006. Consumer-resource body- Pullman. size relationships in common food webs. Blanckenhorn, W.U. 1997. Altitudinal life history Ecology 87: 2411-2417. variation in the dung flies Scathophaga stercoraria and Sepsis cynipsea. Oecologia Burkhard, D.U., Ward, P.I. & Blanckenhorn, 109: 342-352. W.U. 2002. Using age-grading by wing Blanckenhorn, W.U. 2000. The evolution of body injuries to estimate size-dependent adult size: what keeps organisms small? Quart. Rev. survivorship in the field: a case study of the Biol. 75: 385-407. yellow dung fly Scathophaga stercoraria. Ecol. Entomol. 27: 514-520. Blanckenhorn, W.U. 2007. Case studies of the differential equilibrium hypothesis of sexual Calder, W.A. 1984. Size, Function, and Life size dimorphism in dung flies. Pages 106-114 History. Harvard University Press, in: D.J. Fairbairn, W.U. Blanckenhorn, T. Cambridge. Székely, eds. SEX, SIZE AND GENDER ROLES. Cotter, S., Myatt, J., Benskin, C. & Wilson, K. Evolutionary Studies of Sexual Size 2008. Selection for cuticular melanism reveals Dimorphism. Oxford University Press, immune function and life-history trade-offs in Oxford. Spodoptera littoralis. J. Evol. Biol., 21: 1744- Blanckenhorn, W.U. 2009. Causes and 1754. consequences of phenotypic plasticity in body Cox, R.M. & Calsbeek, R. 2009. Sexually size: the case of the yellow dung fly antagonistic selection, sexual dimorphism and Scathophaga stercoraria (Diptera: the resolution of intralocus sexual conflict. Scathophagidae). Pages 369-422 in: D.W. Am. Nat. 173: 176-187. Whitman, T.N. Ananthakrishnan, eds. Endler, J.A. 1986. Natural Selection in the Wild. Phenotypic plasticity of insects: Mechanism Princeton University Press, Princeton. and consequences. Science Publishers, Enfield Fairbairn, D.J. 1997. Allometry for sexual size NH, USA. dimorphism: Pattern and process in the Blanckenhorn, W.U. 2015. Yellow dung fly body coevolution of body size in males and females. size evolution in the field: response to climate Ann. Rev. Ecol. Syst. 28: 659–687. change? Evolution 69: 2227-2234. Fairbairn, D.J. 2005. Allometry for sexual size Blanckenhorn, W.U. & M. Demont. 2004. dimorphism: testing two hypotheses for Bergmann and converse Bergmann latitudinal Rensch’s rule in the water strider Aquarius clines in arthropods: two ends of a continuum? remigis. Am. Nat. 166: S69--84 Integr. Comp. Biol. 44: 413-424. Floate, K.D. & Coghlin, P. 2010. No support for Blanckenhorn, W.U., Kraushaar, U. & Reim, C. fluctuating asymmetry as a biomarker of 2003. Sexual selection on morphological and chemical residues in livestock dung. Can. physiological traits and fluctuating asymmetry Entomol. 152: 354-368. in the yellow dung fly. J. Evol. Biol. 16: 903- Foster, W. 1967. Hormone-mediated nutritional 913. control of sexual behavior in male dung flies. Blanckenhorn, W.U., Fanti, J. & Reim, C. 2007. Science 158: 1596-1597. Size-dependent energy reserves, energy utilization and longevity in the yellow dung González-Santoyo, I. & Córdoba-Aguilar, A. fly. Physiological Entomology 32: 372-381. 2012. Phenoloxidase: a key component of the Blanckenhorn, W.U. & Hosken, D.J. 2003. The insect immune system. Entomo. Exp. Appl. heritability of three condition measures in the 142: 1-16. yellow dung fly. Behav. Ecol. 14: 612-618. Gotanda, K., Correa, C., Turcotte, M.M., Rolshausen, G. & Hendry, A.P. 2015. Linking

12 bioRxiv preprint doi: https://doi.org/10.1101/136325; this version posted January 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Wolf U. Blanckenhorn Selection by a fungal parasite in dung flies

macro-trends and micro-rates: re-evaluating McGregor, R. R. & Roitberg, B.D, 2000. Size- micro-evolutionary support for Cope’s rule. selective oviposition by parasitoids and the Evolution 69: 1345-1354. evolution of life-history timing in hosts: Fixed preferences vs. frequency-dependent host Hammer. O. 1941. Biological and ecological selection. Oikos 89: 305-312. investigations on flies associated with Merilä, J. & Hendry, A.P. 2014. Climate change, pasturing cattle and their excrement. adaptation, and phenotypic plasticity: the Vidensk. Medd. Dansk Naturh. Foren. problem and the evidence. Evol. Appl. 7: 1-14. 105: 140-393. Merilä, J., Sheldon, B.C. & Kruuk, L.E.B. 2001. Honek, A. 1993. Instraspecific variation in body Explaining stasis: microevolutionary studies size and fecundity in insects: a general in natural populations. Genetica 112-113: 199- relationship. Oikos 66: 483-492. 222. Hosken, D.J., Blanckenhorn, W.U. & Ward, P.I. 2000. Møller, A. P. 1993. A fungus infecting domestic Developmental stability in the yellow dung fly flies manipulates sexual behaviour of its host. (Scathophaga stercoraria): fluctuating asymmetry, Behav. Ecol. Sociobiol. 33: 403-407. heterozygosity and environmental stress. J. Evol. Møller, A.P. & Swaddle, J.P. 1997. Asymmetry, Biol. 13: 919–926. Developmental Stability and Evolution. Jann, P., Blanckenhorn, W. U. & Ward, P. I. 2000. Oxford University Press, Oxford. Temporal and microspatial variation in the Møller, A.P. & Thornhill, R. 1997. A meta- intensities of natural and sexual selection in analysis of the heritability of developmental the yellow dung fly Scathophaga stercoraria. stability. J. evol. Biol. 10: 1-16. J. evol. Biol. 13: 927-938. Mousseau, T.A. & Roff, D.A. 1987. Natural Kingsolver, J.G., Hoekstra, H.E., Hoekstra, J.M., selection and the heritability of fitness Berrigan, D., Vignieri, S.N., Hill, C.E., Hoang, components. Heredity 59: 181-197. A., Gibert, P. & Beerli, P. 2001. The strength Nielsen, C. & Hayek, A.E. 2006. Diurnal pattern of phenotypic selection in natural populations. of death and sporulation in Entomophaga Am. Nat. 157: 245-261. maimaiga-infected Lymantria dispar. Kingsolver, J.G. & D.W. Pfennig. 2004. Entomol. Exp. Appl. 118: 237-243. Individual-level selection as a cause of Cope’s rule of phyletic size increase. Evolution Nunn, C.L., Lindenfors, P., Pursall, E.R. & Rolff, 58:1608-1612. J. 2009. On sexual dimorphism in immune function. Phil. Trans. Roy. Soc. Lond. B 364: Knierim, U., Van Dongen, S., Forkman, B., 61-69. Tuyttens, F.A.M., Špinka, M., Campo, J.L., & Palmer, A.R. 2000. Quasi-replication and the Weissengruber, G.E. 2007. Fluctuating contract of error: lessons from sex ratios, asymmetry as an animal welfare indicator — heritabilities and fluctuating asymmetry. Ann. A review of methodology and validity. Physiol Rev. Ecol. Syst. 31: 441-480. Behav. 92: 398-421. Palmer, A. R. & Strobeck, C. 1986. Fluctuating asymmetry: measurement, analysis, patterns. Kraushaar, U. & Blanckenhorn, W.U. 2002. Ann. Rev. Ecol. Syst. 17: 391-421. Population variation in sexual selection and its Parker, G.A. 1970. The reproductive behaviour effect on body size allometry in two species of and the nature of sexual selection in flies with contrasting sexual size dimorphism. Scathophaga stercoraria L. (Diptera: Evolution 56: 307-321. Scathophagidae) II. The fertilization rate and Lande, R. & Arnold, S.J. 1983. The measurement the spatial and temporal relationships of each of selection on correlated characters. Evolution sex around the site of mating and oviposition. 37: 1210-1226. J. Anim. Ecol. 39: 205-228. Liggett, A. C., Harvey, I.F. & Manning, J.T. 1993. Parker, G.A. 1979. Sexual selection and sexual Fluctuating asymmetry in Scathophaga conflict. In: Sexual Selection and Reproductive stercoraria L.: successful males are more Competition in Insects (N. S. Blum & N. A. symmetrical. Anim. Behav. 45: 1041-1043. Blum, eds), pp. 123-166. Academic Press, Maitland, D.P. 1994. A parasitic fungus infecting New York. yellow dungflies manipulates host perching Preziosi, R.F. & Fairbairn, D.J. 2000. Lifetime behaviour. Proc. Roy. Soc. Lond. B 258: 187- selection on adult body size and components 193. of body size in a waterstrider: opposing selection and maintenance of sexual size dimorphism. Evolution 54: 558-566.

13 bioRxiv preprint doi: https://doi.org/10.1101/136325; this version posted January 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Wolf U. Blanckenhorn Selection by a fungal parasite in dung flies

Polak, M. (ed.) 2003. Developmental instability. resistance in the yellow dung fly Scathophaga Causes and consequences. Oxford: Oxford stercoraria. J. evol. Biol. 20: 2192-2199. University Press. Schmidt-Nielsen, K. 1984. Scaling. Why is animal size so important? Cambridge University Rantala, M.J. & Roff, D.A. 2005. An analysis of Press, Cambridge. trade-offs in immune function, body size and Sigurjónsdóttir, H. & Snorrason, S.S. 1995. development time in the Mediterranean field Distribution of male yellow dungflies around cricket, Gryllus bimaculatus. Funct. Ecol. 19: oviposition sites: the effect of body size. Ecol. 323-330. Entomol. 20: 84-90. Rantala, M.J., Koskimäki, J., Taskinen, J., Steenberg, T., Jespersen, J.B., Jensen, K.-M.V., Tynkkynen, K. & Suhonen, J. 2000. Nielsen, B.O. & Humber, R. A. 2001. Immunocompetence, developmental stability Entomopathogenic fungi in flies associated and wing spot size in the damselfly Calopteryx with pastured cattle in Denmark. J. Invert. splendens L. Proc. Roy. Soc. Lond. B 267: Pathol. 77: 186-197. 2453–2457. Steinkraus, D.C. & Kramer, J.P. 1988. Rantala, M.J., Ahtiainen, J.J. & Suhonen, J. 2004. Entomophthora scatophagae, a fungal Fluctuating asymmetry and immune function pathogen of the yellow dung fly, Scatophaga in a field cricket. Oikos 109: 479–484. stercoraria. Mycotaxon 32: 105-113. Stoks, R., Geerts, A.N. & de Meester, L. 2014. Rantala, M.J., Roff, D.A. & Rantala, L.M. 2007. Evolutionary and plastic responses of Forcepts size and immune function in the freshwater invertebrates to climate change: earwig Forficula auricularia. Biol. J. Linn. realized patterns and future potential. Evol. Soc. 90: 509-516. Appl. 7: 42-55. Reim, C., Teuschlm Y, & Blanckenhorn, W.U. Swaddle, J.P. 1997. Developmental stability and 2006. Size-dependent effects of larval and predation success in an insect predator-prey adult food availability on reproductive energy system. Behav. Ecol. 8: 433-436. allocation in the yellow dung fly. Funct. Ecol. Van Dongen, S.2006. Fluctuating asymmetry and 20: 1012-1021. developmental instability in evolutionary biology: past, present and future. J. Evol. Biol. Rensch, B. 1950. Die Abhängigkeit der relativen 19: 1727–43. Sexualdifferenz von der Körpergrösse. Bonn. Zoolog. Beitr. 1: 58-69. Vucic-Pestic, O., Rall, B.C., Kalinkat, G. & Brose, U. 2010. Allometric functional Roff, D.A. 1992. The Evolution of Life Histories. response model: Body masses constrain Chapman & Hall, New York. interaction strengths. J. Anim. Ecol. 79: 249- Rolff, J. & Reynolds, S. 2009. Insect Infection and 256. Immunity: Evolution, Ecology, and Mechanisms. Oxford University Press. Yourth, C.P., Forbes, M. & Smith, B.P. 2002. Schilthuizen, M. & Kellermann, V. 2014. Immune expression in a damselfly is related to Contemporary climate change and terrestrial time of season, not to fluctuating asymmetry invertebrates: evolutionary versus plastic or host size. Ecol. Entomol. 27: 123–128. changes. Evol. Appl. 7: 56–67. Schmid-Hempel, P. 2005. Evolutionary ecology Zuk, M. & Kolluru, G.R. 1998. Exploitation of of insect immune defenses. Annu. Rev. sexual signals by predators and parasitoids. Entomol. 50: 529-551. Quart. Rev. Biol. 73: 415-438. Schwarzenbach, G.A., Hosken, D.J. & Ward, P.I. 2005. Sex and immunity in the yellow dung fly Scathophaga stercoraria. J. evol. Biol. 18: 455-463. Schwarzenbach, G.A. & Ward, P.I. 2006. Responses to selection on phenoloxidase activity in yellow dung flies. Evolution 60: 1612-1621. Schwarzenbach, G.A. & Ward, P.I. 2007. Phenoloxidase activity and pathogen

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