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O R I G I NA L A RT I C L E doi:10.1111/evo.12408

THE INTERPLAY BETWEEN LOCAL ECOLOGY, DIVERGENT SELECTION, AND GENETIC DRIFT IN POPULATION DIVERGENCE OF A SEXUALLY ANTAGONISTIC FEMALE TRAIT 1,5 ¨ Kristina Karlsson Green,1,2,3 Erik I. Svensson,1 Johannes Bergsten,4 Roger Hardling, and Bengt Hansson1 1

¨ Department of Biology, Lund University, Solvegatan 37, SE-223 62 Lund, Sweden

2

Current Address: Department of Biosciences, University of Helsinki, PO Box 65, FI-00014 Helsinki, Finland 3

4

E-mail: [email protected]

Entomology Department, Swedish Museum of Natural History, Box 50007, SE-10405 Stockholm, Sweden

5

¨ Current Address: Cykelvagen 4, SE-24750 Dalby, Sweden

Received November 27, 2013 Accepted March 3, 2014 Genetically polymorphic species offer the possibility to study maintenance of genetic variation and the potential role for genetic drift in population divergence. Indirect inference of the selection regimes operating on polymorphic traits can be achieved by comparing population divergence in neutral genetic markers with population divergence in trait frequencies. Such an approach could further be combined with ecological data to better understand agents of selection. Here, we infer the selective regimes acting on a polymorphic mating trait in an insect group; the dorsal structures (either rough or smooth) of female diving beetles. Our recent work suggests that the rough structures have a sexually antagonistic function in reducing male mating attempts. For two species (Dytiscus lapponicus and Graphoderus zonatus), we could not reject genetic drift as an explanation for population divergence in morph frequencies, whereas for the third (Hygrotus impressopunctatus) we found that divergent selection pulls morph frequencies apart across populations. Furthermore, population morph frequencies in H. impressopunctatus were significantly related to local bioclimatic factors, providing an additional line of evidence for local adaptation in this species. These data, therefore, suggest that local ecological factors and sexual conflict interact over larger spatial scales to shape population divergence in the polymorphism. KEY WORDS:

AFLP, Dytiscidae, frequency-dependent selection, genetic polymorphism, sexual conflict, WorldClim.

How within-population genetic diversity is translated to betweenpopulation divergence and ultimately speciation is a classic research topic in evolutionary biology (see, e.g., Gray and McKinnon 2007; Svensson et al. 2009). Of particular interest are populations or species that are polymorphic with respect to phenotypic traits that affect fitness. Such genetic polymorphisms could be a transient phase when an allele, or a set of alleles, sweeps through a population for a limited time until the alleles are fixed and genetic uniformity is reached in the population again (Ford 1945). There are, however, many examples of balanced polymorphisms, meaning that multiple alleles are maintained within the population at a more or less stable genetic equilibrium (Ford 1945). Such polymorphisms below the species level are C

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quite common in nature and have been documented in a wide variety of animal taxa, including insects (Losey et al. 1997; Karlsson et al. 2008), lizards (Calsbeek et al. 2010; Huyghe et al. 2010), spiders (Gillespie and Oxford 1998; Oxford 2005), and birds (Roulin 2004; Pryke and Griffith 2007). Polymorphisms are generally thought to be maintained by some form of balancing selection (Svensson et al. 2009; Ibarra and Reader 2013), usually in the form of frequency-dependent selection or overdominant selection (heterozygote advantage; Hedrick 2007), where frequency-dependent selection may be more powerful in maintaining polymorphisms (Hedrick 1972; Clarke 1979). In particular, negative frequency-dependent selection, where the rare morph has an advantage, has gained

C 2014 The Society for the Study of Evolution. 2014 The Author(s). Evolution Evolution 68-7: 1934–1946

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much theoretical interest but surprisingly few rigorous empirical studies have demonstrated the evolutionary maintenance of phenotypic polymorphisms in natural settings (see, e.g., Sinervo and Lively 1996; Svensson et al. 2005; discussed in Ibarra and Reader 2013). Genetic drift might also partly affect population divergence of morph frequencies in natural populations, although the effects of drift are usually even more difficult to demonstrate convincingly (Oxford 2005; Runemark et al. 2010). Sex-limited polymorphism is a special case of genetic polymorphism, where multiple morphs are only expressed in either males or females, but not in both sexes (Kunte 2009; Svensson et al. 2009). Female mating polymorphisms have been described in a few cases, and could potentially be common in species experiencing sexual conflict (Svensson et al. 2009). One example of sexual conflict affected female polymorphism comes from damselflies of the genus Ischnura, where several species have two or three female color morphs, thought to coexist through frequency-dependent sexual antagonism (Robertson 1985; Svensson et al. 2005; Gosden and Svensson 2009). Another recently described example of female polymorphism (or dimorphism) that is likely associated with sexual conflict occurs in diving beetles (Coleoptera: Dytiscidae). Females of several diving beetle species have rough structures on their elytron, for example, furrows or granules (Nilsson and Holmen 1995). These rough structures have been suggested to be an important trait in sexual conflict (Bergsten et al. 2001; Miller 2003; Bergsten and Miller 2007) and our recent experimental work confirms the sexually antagonistic function of these rough structures, as males experience difficulties in adhering to the rough surfaces (Karlsson Green et al. 2013). However, in some species, two different female morphs coexist locally within populations; one with smooth elytron and the other with rough elytron (Nilsson and Holmen 1995; Karlsson Green et al. 2013). Recent theory suggests that sexual conflict can favor the emergence of genetic mating polymorphisms in both males and females (Gavrilets and Waxman 2002), and negative frequency-dependent selection could subsequently maintain multiple morphs in both sexes (Härdling and Bergsten 2006; Härdling and Karlsson 2009). To date, however, the selective regimes maintaining the female morphs among diving beetles in local populations have not been thoroughly investigated across natural populations. Indirect inferences of selection regimes are usually performed by comparing the degree of genetic divergence between populations (FST values) of traits that are assumed to be neutral with those traits that are suspected to be target of selection (Merilä and Crnokrak 2001; Whitlock 2008). If FST values for the selected loci exceed FST for the neutral loci, the populations show greater differentiation in the selected loci than is expected by genetic drift alone. Such a discrepancy suggests that divergent selection caused by different local environments has pulled pop-

ulations apart in their phenotypes, for example, by favoring local adaptations in the selected traits. Conversely, if the FST values for the selected loci are lower than FST for the neutral loci, populations are less differentiated from each other than expected by genetic drift. Such a finding suggests stabilizing selection and that similar selection pressures might operate across all populations, preventing population divergence in morph frequencies (Merilä and Crnokrak 2001; Whitlock 2008). Finally, if the FST (selected) would equal the FST (neutral), population differentiation in the (presumed) adaptive traits does not differ significantly from what is expected from genetic drift alone. A failure to reject genetic drift could imply that drift does indeed operate on the trait in focus. Alternatively, both stabilizing and diversifying selection might operate on the trait, but in opposite directions, that is, a dynamic selective balance. Of particular concern when investigators do not find any difference between neutral markers and adaptive trait divergence using these indirect inferences is the low statistical power of this approach (Whitlock 2008). This is important if one aims to find indirect evidence for stabilizing selection and expects less population differentiation than expected from drift (Beaumont and Nichols 1996; Whitlock 2008). Neutral FST values might often be very low (or even close to zero), particularly among newly established populations or species with high dispersal capacity, such as many insect species, like odonates (Svensson et al. 2004; Abbott et al. 2008). In such systems, it might be extremely difficult to get FST values for the adaptive traits that are even lower, as FST can (per definition) never be less than zero (Abbott et al. 2008; Whitlock 2008). In spite of these problems and limitations, several recent studies have successfully used such an FST comparison to infer the selection pressures operating in natural populations (Merilä and Crnokrak 2001; McKay and Latta 2002). Examples of recent studies using indirect inferences include ecotype divergence (Manier et al. 2007; Eroukhmanoff et al. 2009) and population divergence of color morph frequencies (Abbott et al. 2008; Sanchez-Guillen et al. 2011; Cox and Rabosky 2013). Nevertheless, the low statistical power of such indirect inferences of selection has also led some researchers to suggest that this approach is not the only step in investigations of how selection influences population divergence (Whitlock 2008). A full understanding of the ecological agents and causes of selection would also require information about environmental drivers of the observed population divergence, according to Whitlock (2008). Here, we investigate and discuss the nature of the selective forces operating on female elytral polymorphism in diving beetles. We compared neutral genetic FST estimates with FST estimates of this female mating polymorphism (female morph frequencies) and performed such studies on three different species and genera of diving beetles: Dytiscus lapponicus (Gyllenhal), Graphoderus zonatus (Hoppe), and Hygrotus impressopunctatus (Schaller).

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If stabilizing selection arising from frequency-dependent sexual conflict maintains the female polymorphism in these species, we would expect to find FST (selected) < FST (neutral). In contrast, if divergent selection due to different local ecological environments is a predominating evolutionary force and results in local adaptation, we would expect FST (selected) > FST (neutral). To further understand the ecological causes behind the potential selection pressures, and how they influence local adaptation for this polymorphic and sexually antagonistic mating trait in females, we analyzed the effect of local microclimatic variables and how they were related to local morph frequencies using dissimilarity-based analyses (Goslee and Urban 2007). We end with a critical discussion about the problems and pitfalls of using indirect inferences about selection to infer the selective regimes on suspected adaptive traits in polymorphic systems, and call for closer integration between ecology and sexual selection, in line with recent suggestions (Svensson and Waller 2013; Miller and Svensson 2014).

Material and Methods STUDY SPECIES AND FIELDWORK

Diving beetles (Dytiscidae) are a global family found mainly in fresh waters but with some species also in brackish waters (Nilsson and Holmen 1995). Adults are aquatic but may leave the water for migration or overwintering (Nilsson and Holmen 1995). Male diving beetles commonly have adhesive setae on their fore and middle legs (Nilsson and Holmen 1995). In some species, these setae are formed as symmetrical suction cups and used for capturing and holding females during mating (Wichard et al. 2002). Females of many diving beetle species have structural modifications on their elytra; for example, deep furrows or granules. However, some diving beetle species are polymorphic with both a “rough” and a “smooth” female morph. Mating is characterized by an intense precopulatory struggle between the sexes and also by postcopulatory mate-guarding (Aiken 1992; Wichard et al. 2002). A sexual conflict over mating has thus been proposed (Bergsten et al. 2001; Miller 2003), and this hypothesis is supported by the findings from comparative phylogenetic studies of the male and female antagonistic traits (Bergsten and Miller 2007) and from recent adhesion experiments that have revealed that males have more difficulties in adhering to the rough than the smooth female morph (Karlsson Green et al. 2013). The frequencies of these female morphs vary between populations (Bergsten et al. 2001; Bilton et al. 2008; Drotz et al. 2010), which sometimes have resulted in a pronounced geographic pattern of morph frequencies (Nilsson and Holmen 1995; Bilton et al. 2008; Drotz et al. 2010). We conducted our study on three species of diving beetles: D. lapponicus, G. zonatus, and H. impressopunctatus (Fig. 1). D. lapponicus (Fig. 1A) is a large diving beetle, 24.1–30.0 mm in size, which is present from Great Britain and Fennoscandia

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Diving beetle species used in the study: (A) Dytiscus lapponicus, (B) Graphoderus zonatus, and (C) Hygrotus impres-

Figure 1.

sopunctatus. From left to right in each figure: male, rough female morph, smooth female morph. Scales to the right of each beetle indicate 1 mm.

to West Siberia (Nilsson and Holmen 1995). Females are either smooth or with 10 longitudinal furrows on the elytron, and both these morphs coexist in Sweden (Nilsson and Holmen 1995). G. zonatus (Fig. 1B) is medium-sized, about 12.0–15.7 mm long, and widespread from central Fennoscandia to Siberia. In this species, females are either smooth or granulate (Nilsson and Holmen 1995). Both morphs coexist in central and northern Fennoscandia, but the granulate morph does not exist in southern Sweden (Nilsson 1986). Finally, H. impressopunctatus (Fig. 1C) is a small diving beetle with a body length of 4.1–5.5 mm (Nilsson and Holmen 1995). The species has a holarctic distribution and is present both in North America and in Europe, from the Iberian Penninsula to Fennoscandia and eastwards to northern China and Japan (Nilsson and Holmen 1995). H. impressopunctatus is found both in freshwater and in brackish waters along the Baltic Sea coast line (Nilsson and Holmen 1995). Females are either smooth and shiny or matt and microreticulate, and the two forms are present in both the palearctic and nearctic (Nilsson and Holmen 1995;


Larson et al. 2000). In contrast to D. lapponicus and G. zonatus, both males and females have adhesive suction cup like setae on their fore and middle legs in H. impressopunctatus (J. Bergsten, pers. obs.). However, the adhesive setae are of two types and their numbers and distribution differ markedly between the sexes. Males have broader feet segments with a larger surface of setae which, in contrast to females, is differentiated into large suction cups in the middle and smaller adhesive setae at the lateral edges (J. Bergsten, pers. obs.). In all three species, the two different female morphs coexist in Sweden (and for H. impressopunctatus also in Denmark) and no intermediate forms have been found. We collected diving beetles from Sweden (D. lapponicus, G. zonatus, and H. impressopunctatus) and Denmark (H. impressopunctatus) during Autumn 2007 and 2008 (Fig. 2, Table 1). Beetles were captured with traps, which were baited with fish and left over night, or by netting along the shore line of ponds. Individuals used in the genetic study were immediately preserved in 96% ethanol. The population frequencies of the female morphs were calculated from field samples consisting of 10–117 females (Table 1). We used χ2 -statistics (Statsoft 2004) to test if the morph frequencies differed among populations within each species. ASSUMPTION OF MORPH INHERITANCE

In each of our study species, only two distinct morphs are found and no intermediate forms exist (Nilsson and Holmen 1995). Controlled breeding experiments on other species of diving beetles have suggested a Mendelian inheritance of a major locus, where the rough morph allele is dominant over the smooth morph allele (Inoda et al. 2012). In this study, we therefore assume a one-locus two-allele system where the rough females are either homozygotes or heterozygotes, and all the smooth females are recessive homozygotes. LABORATORY WORK

We extracted DNA from the head of the beetles with a standard phenol–chloroform protocol (Sambrook et al. 1989). Our samples were analyzed with amplified fragment length polymorphism (AFLP) as described by Vos et al. (1995) with the minor modifications described in Bensch et al. (2002). The EcoRI primers (E primers) were 5 -end labeled with a fluorescent dye (FAM, HEX, or NED; Applied Biosystems, Foster City, CA). Three primer combinations were analyzed together with a Genescan 500 (ROX) size standard in an ABI 3730 capillary sequencer (Applied Biosystems). We tested six different primer combinations and selected four of them for further analysis: Mcga-Etga, Mcgg-Etga, Mcgc-Etcg, and McatEtcg. Every primer combination was screened for polymorphic fragments that had sizes in the range of 50 bp up to about 350 bp with Genemapper version 4.0 (Applied Biosystems). We extracted polymorphic loci with the semiautomated method

Maps over the sampled populations from each species. Each panel displays the natural morph frequency illustrated in pie charts for each sampled population (black = proportion of the

Figure 2.

rough morph, white = proportion of the smooth morph). The Xs on the lower magnitude maps over Sweden and Denmark within each panel denote all populations used in the study but only the populations with pie charts where sampled for a particular species. (A) D. lapponicus populations, (B) G. zonatus populations, and (C) H. impressopunctatus populations.

presented by Whitlock et al. (2008), using the script AFLPScore (http://www.sheffield.ac.uk/molecol/software/aflpscore.html) in R (R Development Core Team 2010). The AFLPScore analyses resulted in many loci being represented by very rare or common polymorphisms. Because structure analyses based

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The number of individuals from each population and species used in the AFLP analyses, the morph frequency (number of smooth females/total number of females) in these populations as well as the number of females used for calculating morph frequency.

Table 1.

Species

Population

No. of Inds. for AFLP

Morph Frequency

Total No. of Females

Dytiscus lapponicus

Lomtjärn L˚angvikskatan ¨ Oster-Skivsj¨ on Mjösjön Fjällmyran Lomtjärn L˚angvikskatan ¨ Oster-Skivsj¨ on Mjösjön ∗ Storkvarnhustjärnen ∗ ¨ Oltertjärnarna Fjällmyran Bottorp ∗ Ishöj Strand Kulltorpsdamm Mellbyören Norra Sandby ∗ Revingehed Svinö

8 14 12 16 15 18 13 14 15 10 18 18 16 17 16 15 16 8 7

0.12 0.29 0.35 0.57 0.1 0.57 0.88 0.55 0.78 0.7 0.34 0.76 0.3 0.89 0.22 0.08 0.02 0.87 0

17 17 40 23 10 76 60 33 18 33 38 25 117 18 32 36 33 23 34

Graphoderus zonatus

Hygrotus impressopunctatus

∗

Population excluded in an additional analysis involving only a subset of the populations.

on such data will have low power, we excluded loci with rare or common alleles (cutoff: bands present or absent in less than 14% of the individuals). Furthermore, for some loci there was a batch effect with unequal number of bands being amplified in different PCRs (96-plates). To detect and remove loci affected by such batch effects, we conducted χ2 -tests over plates for each locus and loci with P < 0.001 were removed. From each population, we initially included 20 individuals in the genetic study. However, the number of amplified AFLP loci may differ between individuals due to variation in template DNA and PCR quality. Therefore, we removed individuals with suspiciously few bands (cutoff: 30% of the total number of loci) or many bands (cutoff: 40%), and after this reduction 7–18 individuals per population (about 70% of the total sample) remained (Table 1). In our analyses, we used AFLP genotypes at 253 loci in 65 D. lapponicus individuals from five populations, at 285 loci in 106 G. zonatus individuals from seven populations, and at 223 loci in 95 H. impressopunctatus individuals from seven populations (Fig. 2, Table 1).

PAIRWISE FST VALUES

AFLP markers represent a random sample of loci in the genome, of which the vast majority are likely to be noncoding and neutral. Pairwise FST values, both for the neutral genetic data and the morph frequencies, were calculated using the software Arlequin 3.1 (Excoffier et al. 2005). Our data were organized to Arlequin

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input files with the R-script AFLPdat (Ehrich 2006). We used 10,000 permutations for FST calculations. Arlequin is a program initially developed for analysis of codominant data and does not take the dominance of AFLP into account when estimating allele frequencies. This introduces an underestimation of the frequency of the recessive allele that is nonlinear with respect to actual allele frequencies, which might lead to slightly overestimated FST values. Despite this, and perhaps indicating that the bias might be relatively minor, it is common to use Arlequin for analysis of ˚ AFLP data (see Bensch and Akesson 2005 and references therein). We also analyzed our data with the program AFLPsurv, which is developed for AFLP data and does account for dominance when estimating allele frequencies. In AFLPsurv, we obtained pairwise FST values while using a Bayesian method with uniform prior distribution for estimating the allele frequencies and by assuming Hardy–Weinberg proportions of the data. We allowed 10,000 permutations for calculating the FST values.

FST COMPARISONS

As FST (per definition) cannot be negative, all negative estimates were pooled to 0 in the statistical analyses. We compared our FST values between all pairs of populations for the morphological trait and the neutral AFLP data using paired t-tests for each species. As the data points are interdependent, we used resampling statistics (50,000 permutations) with the software Resampling Stats (Simon 2000). Recently, this method of comparing the difference in mean


of FST values has been criticized and it has instead been suggested that a comparison of the FST value of the focal trait with the whole distribution of neutral FST values is more appropriate (Whitlock 2008). We thus also compared our FST (morph) values with the entire FST (neutral) distribution. To obtain the overall FST distribution, we used the option for finding loci under selection (FST outlier analysis) in Arlequin 3.5.1 (Excoffier and Lischer 2010). In this analysis we used the Hierarchical island model and allowed for 50,000 permutations. For each species, we treated the populations as one group and set the number of demes as the number of sampled populations. We also analyzed the populations divided into several groups depending on geographic location, but as the group division did not affect the results we do not present these results here. This analysis plots the FST values for each locus against the observed heterozygosity. We then compared the FST (morph), both with the entire FST (neutral) distribution, and we also compared the predicted value from the neutral distribution only with the expected level of heterozygosity of the morph locus. We performed all FST analyses for all populations within each species. We followed this up with a more restricted comparison where a smaller number of geographically very distant populations were removed from G. zonatus and H. impressopunctatus. The logic behind this was to remove as much ecological differences as possible between populations to minimize the effect of divergent selection and to increase the possibility of detecting stabilizing selection. We were also interested in comparing population divergence of the different species when the geographic distances between the populations were more similar to better understand the causes of any interspecific differences. In these analyses, the populations Ishöj Strand and Revingehed were, therefore, removed from the H. impressopunctatus dataset, ¨ and the populations Oltertj¨ arnarna and Storkvarnhustjärnen were removed from the G. zonatus dataset (see ∗-labeled populations in Table 1). ISOLATION BY DISTANCE

We investigated if there was any pattern of isolation by distance using Mantel test in Arlequin 3.1 (Excoffier et al. 2005) for each species. We performed this test using two settings. First, we included all populations from each species. Second, we only used a smaller subset of populations for G. zonatus and H. impressopunctatus (the same populations removed as above). ENVIRONMENTAL ANALYSES

To address the potential for divergent selection and the effect of local environmental conditions on the morph frequencies, we used the ecodist package (Goslee and Urban 2007) in R (R Development Core Team 2010) to analyze the correlations between local morph frequencies and different temperature and precipita-

tion variables with Mantel tests. We chose temperature variables, as temperature has well-recognized effects on the fitness of most insects (e.g., Deutsch et al. 2008), and precipitation variables, as precipitation might be important for those species that inhabit small and temporary water bodies, such as H. impressopunctatus. For each environmental variable, we performed both a Mantel test and a partial Mantel test, where the environmental variable matrix was analyzed together with a distance matrix. Environmental variables were obtained from the WorldClim database (Hijmans et al. 2005), which is a collection of various bioclimatic variables and available online at http://www.worldclim.org.

Results The morph frequencies differed significantly between the populations within each of the three species (D. lapponicus: χ2 = 77.17, df = 4, P < 0.001; G. zonatus: χ2 = 215.33, df = 6, P < 0.001; H. impressopunctatus: χ2 = 354.36, df = 6, P < 0.001). The pairwise FST values for both neutral loci and the morph locus obtained using both Arlequin and AFLPsurv are presented in Supporting Information Tables S1–S5. The only species where a significant difference between FST (neutral) and FST (morph) was found was H. impressopunctatus. In this species, FST (morph) exceeded FST (neutral) both in the analysis based on Arlequin FST s and on AFLPsurv FST s. This result remained significant when analyzed with resampling and was found in both the analysis of all populations (Arlequin FST : t = 4.57, df = 20, P = 0.004; AFLPsurv FST : t = 5.05, df = 20, P = 0.002) and where only a subset of populations were included (Arlequin FST : t = 1.97, df = 9, P = 0.036; AFLPsurv FST : t = 2.53, df = 9, P = 0.006; P-values from resampling analyses). When comparing the FST values obtained with Arlequin and AFLPsurv, respectively, half of the comparison showed significant differences (Supporting Information Table S6), and, curiously enough, all significant differences were obtained in analyses of the morph FST s. In all these comparisons, the FST values obtained with Arlequin were higher than the FST s obtained with AFLPsurv (Supporting Information Table S6). When FST (morph) was compared to the FST (neutral) in the outlier analysis, the FST (morph) values for both G. zonatus and D. lapponicus fell within the range of the neutral genetic variation (Table 2). In contrast, for H. impressopunctatus FST (morph) once again exceeded the FST (neutral) distribution when all seven populations where analyzed, whereas it fell within the tail of neutral variation when only the more geographically restricted population subset was analyzed (Table 2; Figs. 3, 4). These results were obtained both when the FST (morph) was compared to the entire FST distribution (Fig. 4) and with the distribution at the corresponding level of heterozygosity (Fig. 3).

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FST values for selected trait (FST [Morph]), the observed heterozygosity of the morph locus, and the FST values for neutral variation (FST [Neutral] for the 0.05 and 0.95 quantile) at approximately the same level of heterozygosity as the morph locus.

Table 2.

Species (Analysis)

Morph Heterozygosity

Neutral Heterozygosity

FST (Morph)

FST (Neutral) 0.05 Quantile

FST (Neutral) 0.95 Quantile

D. lapponicus G. zonatus (all populations) G. zonatus (reduced subset) H. impressopunctatus (all populations) H. impressopunctatus (reduced subset)

0.453 0.469 0.439 0.441 0.309

0.438 0.475 0.447 0.450 0.310

0.097 0.132 0.103 0.399 0.120

0.018 0.008 0.008 0.008 0.008

0.433 0.198 0.218 0.163 0.200

A bold value indicates an FST (morph) value that falls outside the range of FST (neutral) values.

Distribution of mean pairwise FST values for all neutral loci (grey bars) and the mean pairwise FST values for the assumed morph locus (black bar) for H. impressopunctatus (analysis includ-

Figure 4.

ing all populations). Negative FST s were pooled to 0 in all statistical analyses.

Figure 3. FST values plotted against the observed heterozygosity for H. impressopunctatus. Open circles indicate FST (neutral) val-

ues. Filled black dots are FST (morph) values. Dotted lines indicate the 0.05 and 0.95 quantiles for the distribution of FST (neutral) and solid lines indicate the 0.5 quantile. (A) Analysis where all seven populations of H. impressopunctatus are included. FST (morph) falls outside the distribution of FST (neutral). (B) Analysis where only five populations of H. impressopunctatus are included. FST (morph) fall within the distribution of FST (neutral). Note that a few of the AFLP markers also fall outside the neutral zone, indicating that these molecular markers are either nonneutral or are linked to loci under selection.

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Based on these results, we cannot conclusively reject genetic drift as an explanation for population divergence in morph frequencies of either G. zonatus or D. lapponicus. For H. impressopunctatus, by contrast, our results indicate diversifying selection when the entire range of populations is included, whereas drift could not be excluded when we analyzed a more limited subset of populations over the smaller geographical area. None of the regressions between FST (neutral) and FST (morph) were statistically significant (data not shown). Moreover, in all outlier analyses a small number of AFLP loci fell outside the upper 5% quantile. This indicates that these loci are under selection, or linked to loci under selection, and that all these AFLP markers are strictly not neutral. As AFLP markers represent a random sample of loci, more or less randomly spread over the genome, it is not surprising that a few markers appeared to be nonneutral (Fig. 3). All Mantel tests of isolation by distance


were found to be nonsignificant, both for the neutral markers and the morph frequencies. In the environmental analyses of microclimatic variables, we only found significant relationship between population morph frequencies and the environmental variable in H. impressopunctatus, which was also the species where the molecular data allowed us to reject genetic drift as an explanation for population divergence in morph frequencies (see above). For this species, a number of both temperature and precipitation variables had significant effects on local morph frequencies (Table 3). Most of these environmental variables were significant when analyzed with the Mantel tests whereas when performing partial Mantel tests, and thus correcting for geographical distance between populations, a subset of these variables remained significant. These variables were “temperature seasonality,” “temperature annual range,” “precipitation of the wettest quarter” as well as “precipitation of the warmest quarter.” In addition, “annual precipitation” and “precipitation of the driest quarter” were nearly significant (Table 3).

Discussion Here, we have used both an indirect molecular approach and a more direct correlative approach involving analysis of environmental variables to infer the selective regimes acting on a sexually antagonistic polymorphic trait: the dorsal dimorphism of female diving beetles. Among our three study species, we found evidence for divergent selection in H. impressopunctatus, whereas for G. zonatus and D. lapponicus we cannot exclude genetic drift as entirely driving population divergence in morph frequencies. In none of the three species, did we find any significant evidence for stabilizing frequency-dependent selection, which should manifest itself as a pattern of lower population divergence in morph frequencies, compared to the neutral markers (Whitlock 2008). The main cause for maintenance of polymorphism within populations has been suggested to be frequency-dependent selection (e.g., Hedrick 1972; Clarke 1979; Hedrick 2007 but see Ibarra and Reader 2013). Under negative frequencydependent selection, rare morphs have a selective advantage. Such frequency-dependent fitness of female morphs might arise under sexual conflict if the most common morph is more involved in mating interactions that lower female fitness (Gavrilets and Waxman 2002). Maintenance of genetic mating polymorphisms due to frequency-dependent sexual conflict has been investigated both theoretically (Härdling and Bergsten 2006; Härdling and Karlsson 2009) and in empirical field studies (Gosden and Svensson 2009; Takahashi et al. 2010; Iserbyt et al. 2013). Comparative and experimental data suggest that these dorsal modifications of female diving beetles are sexually antagonistic traits that benefit females by increasing her control during mating (Bergsten and Miller 2007; Karlsson Green et al. 2013). From the

perspective of sexual conflict, it is thus likely that this morph trait to some extent might be target for negative frequency-dependent selection caused by sexual antagonism. We were, however, unable to detect stabilizing selection in any of our studied species; instead we found significant evidence for divergent selection in H. impressopunctatus. This was further supported by our analyses of how local environmental variables (precipitation and temperature) affected population morph frequencies. These environmental factors might affect the female morphs directly, if they differ in their physiological thermal sensitivity or sensitivity to precipitation. Alternatively, the environmental factors might affect female fitness more indirectly, by altering local sexual conflict dynamics and the relative fitness of the two morphs, which in turn would affect population morph frequencies. However, it is also possible that in this species, sexual conflict is not as pronounced as in the other two studied species, which could tip the balance between sexual conflict and ecological selection in favor of the latter. This is supported by observations of the mating duration that can last for hours in larger species like Dytiscus (Aiken 1992), presumably at a substantial cost, whereas in smaller species like Hygrotus it is much shorter (Miller 2003). Another indication of that the dorsal modifications are target of natural selection besides sexual selection is that several diving beetle species show strong geographic clines in the frequencies of rough females (Nilsson and Holmen 1995; Bilton et al. 2008). In at least one species of diving beetles (Agabus bipustulatus), rough elytral structures are found in both sexes and are more frequent at higher altitudes (Drotz et al. 2010). The rough elytral structures in this species have been hypothesized to serve as protection from the dangerous UV radiation at higher altitudes (Drotz et al. 2010). As a parallel to another system, Cooper (2010) found that color polymorphism and sexual dimorphism in a damselfly varied along an elevation cline, probably as a consequence of red coloration being more UV protective. Although it is not clear whether the rough diving beetle structures actually protect against UV light, the geographic pattern in morph abundance indicates that the elytral structures are also shaped by local environmental factors. However, it still remains to be explained why this polymorphism in most species is sex-limited in expression. An interaction between some ecological factor(s) and sexual conflict is therefore likely. For instance, the relative amount and outcome of mating interactions for the different female morphs might vary with ecological conditions, just like it is increasingly appreciated that local sexual selection regimes are highly environment-dependent and often extremely dynamic (Gosden and Svensson 2008; Cornwallis and Uller 2010; Karlsson et al. 2010; Svensson and Waller 2013; Miller and Svensson 2014). Thus, we would certainly not claim that natural selection and sexual conflict would necessarily exclude each other, but it is highly likely that local ecology interacts with sexual conflict. Multiple and opposing selection

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Table 3.

The effect of local environmental conditions on morph frequencies in H. impressopuctatus.

Environmental Variable Temperature variables Annual mean temperature Temperature range [mean of monthly (max temp − min temp)] Isothermality Temperature seasonality Max temperature of the warmest month Min temperature of the coldest month Temperature annual range Mean temperature of the wettest quarter Mean temperature of the driest quarter Mean temperature of the warmest quarter Mean temperature of the coldest quarter Precipitation variables Annual precipitation Precipitation of the wettest month Precipitation of the driest month Precipitation seasonality Precipitation of the wettest quarter Precipitation of the driest quarter Precipitation of the warmest quarter Precipitation of the coldest quarter

Partial Mantel Test

Mantel Test

r = −0.669 P = 0.991 r = −0.004 P = 0.494 r = −0.207 P = 0.789 r = 0.622 P = 0.019 r = 0.263 P = 0.288 r = −0.504 P = 0.966 r = 0.504 P = 0.032 r = −0.202 P = 0.736 r = −0.650 P = 0.991 r = −0.123 P = 0.589 r = −0.631 P = 0.989

r = 0.508 P = 0.014 r = 0.055 P = 0.424 r = 0.760 P = 0.006 r = 0.954 P = 0.004 r = 0.488 P = 0.028 r = 0.796 P = 0.007 r = 0.925 P = 0.013 r = 0.522 P = 0.052 r = 0.716 P = 0.007 r = 0.527 P = 0.037 r = 0.820 P = 0.008

r = 0.414 P = 0.054 r = 0.329 P = 0.116 r = 0.318 P = 0.182 r = −0.085 P = 0.445 r = 0.453 P = 0.014 r = 0.397 P = 0.072 r = 0.462 P = 0.038 r = 0.236 P = 0.281

r = 0.862 P = 0.008 r = 0.815 P = 0.008 r = 0.568 P = 0.011 r = 0.698 P = 0.013 r = 0.874 P = 0.007 r = 0.861 P = 0.014 r = 0.907 P = 0.010 r = 0.414 P = 0.055

Results from partial Mantel correlations (with geographic distances between populations included) and ordinary Mantel correlations. Environmental variables concern temperature and precipitation and were obtained from the WorldClim database (Hijmans et al. 2005). Significant results are highlighted in bold.

pressures are therefore likely to operate on this polymorphism, just like both natural and sexual selection typically operate on most secondary sexual traits in males (Svensson and Gosden 2007). The role for genetic drift at the phenotypic level is more controversial. Genetic drift has nevertheless been suggested to play some role, albeit minor, in the population divergence of genetic color polymorphism in the spider Enoplognatha ovata (Oxford 2005), in the island lizard Podarcis gaigae (Runemark

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et al. 2010), and in Ischnura damselflies (Sanchez-Guillen et al. 2011). In spiders, for instance, it has been suggested that genetic drift might be the major factor affecting morph frequencies at normal frequencies, and that natural selection only operates when the population morph frequencies enter very extreme values (Oxford 2005). The results in this study might suggest that genetic drift might, at least partly, influence population divergence in the local morph frequencies of two of these diving beetle species.


The effect of genetic drift is expected to be more pronounced in smaller populations and in populations with low levels of gene flow (Christensen 2008). Unfortunately, we do not have any data on either the genetically effective population sizes in these species or reliable estimates of the degree of gene flow between populations. However, in this context it is interesting to note that D. lapponicus is reported to usually have degenerated flight muscles and G. zonatus to be probably flightless (Nilsson and Holmen 1995). This should surely decrease the degree of migration and decrease gene flow between populations of these species. Consistent with this, D. lapponicus also had higher FST values for the neutral markers than the other two species (Supporting Information Table S3). That we were unable to reject a role for genetic drift in two of our three studied species does not necessarily mean that genetic drift is the only factor affecting the morph frequencies in these two species. Failure to reject genetic drift may be due to the counteracting forces of stabilizing and diversifying selection pressures that might outweigh each other in our system (Hansen 1997). It should be noted that the statistical power to reject genetic drift in favor of stabilizing selection is usually considerably lower than the power to detect divergent selection using these indirect approaches to infer selection (Whitlock 2008). This is because the range and the 95 confidence intervals of neutral FST s often approach zero (Beaumont and Nichols 1996; Excoffier et al. 2009), precluding any attempt to reject the null hypothesis. Indirect inferences of selection regimes through FST comparisons are certainly valuable in generating testable hypotheses, and these approaches have been widely used in the past by many evolutionary biologists. Recently, however, Whitlock (2008) raised several important critical concerns regarding the problems of indirect inferences about selection. He cautioned against the common practice of comparing average FST values, and instead advocated more conservative tests where one compared the adaptive trait distribution against the whole distribution of neutral FST s (Whitlock 2008). Here, we chose to compare the FST s both with a traditional test and using the approach suggested by Whitlock (2008). Furthermore, we compared the morph FST values with the distribution of neutral FST values only at a given level of heterozygosity. This last test is probably the most statistically conservative of the three approaches, and it was possible to achieve as we obtained a large number of polymorphic loci through our AFLP analyses. It should further be noticed that many studies based on AFLP markers use Arlequin to analyze the data. However, Arlequin was initially developed for analyzing codominant data and does not take the dominant nature of AFLP data into account when estimating allele frequencies. This introduces an underestimation of the frequency of the recessive allele, which is nonlinear with respect to actual allele frequencies. This in turn will lead to overestimated

FST values, which may affect the analyses and the conclusions. Here, we therefore compared the FST s obtained with Arlequin with FST values obtained with the program AFLPSurv, which is designated for dominant data. In many cases, although not all, the FST s differed significantly between the analyses. In all cases the FST values obtained with Arlequin were larger than the FST s calculated by AFLPsurv. These differences between softwares should be taken under consideration when designing studies and evaluating the risk of overestimating the degree of molecular population differentiation. We obtained similar results in all comparisons, which should make our general conclusions robust. Nevertheless, results from such comparisons between neutral markers and adaptive traits should be interpreted with caution, and should ideally be combined with other approaches, such as ecological information about putative selective factors driving selection (Table 3), natural history considerations, or experiments. As an example of the challenges in understanding selection using these approaches, it is worth pointing to a recent study on polymorphic damselflies (Abbott et al. 2008). In their study, Abbott et al. (2008) used indirect inferences to detect selection in two subsequent years. They found that the inferred selection regimes differed significantly between different seasons. In the first year, they found significant evidence for divergent selection, whereas only two years later, they found significant evidence for stabilizing selection in the same set of populations (Abbott et al. 2008). They interpreted this as evidence for the gradual approach toward a common morph frequency equilibrium set by negative frequency-dependent selection that pulled populations together over time (Abbott et al. 2008). For a thorough understanding of the mechanisms responsible for maintaining a polymorphism, as in the diving beetles, future longitudinal studies within carefully chosen populations would be needed. For newly established populations, or species with high migration between population and habitats, the FST values for neutral markers are likely to differ substantially between years, particularly if there is a high population turnover and frequent extinctions and recolonizations (Ingvarsson et al. 1997; Abbott et al. 2008). Populations are also likely to differ in their selective regimes depending on whether they are in the edges of a species range or in the centre of the species range (Kirkpatrick and Barton 1997; Gosden et al. 2011). Thus, the populations chosen for comparison and the geographical scale of study might greatly affect the results and the interpretations. To maximize the statistical power to detect a significant effect of stabilizing selection on morph frequencies, populations should therefore be chosen as to minimize the effect of local adaptation and drift. One common expectation from stabilizing selection operating on a set of populations is a nonlinear increase in phenotypic population differentiation across generations (Hansen 1997). When a set of populations are diverging from each other

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and are approaching their local adaptive peaks, one would first expect to see a rapid increase in phenotypic diversification, followed by a decrease, and finally the attainment of a plateau (Hansen 1997). The plateau is reached when populations have reached their local adaptive peaks. When populations have approached their adaptive peaks, stabilizing selection maintains their average phenotypes at, or close to, their phenotypic optima. Statistically speaking, this is equal to a so-called Ohrnstein–Uhlenbeck process of phenotypic diversification (Hansen 1997). In this model, stabilizing selection creates a “rubber-band” effect that prevents populations from diverging further from each other (Hansen 1997). Although this model was primarily motivated to explain patterns of population divergence for continuous phenotypic traits, it is also applicable to discrete phenotypic traits, like the diving beetle traits. If these elytral structures are subject to negative frequencydependent (stabilizing) selection, morph frequencies will become stable over a large geographic area, unless this stabilizing effect is counteracted by other evolutionary forces, such as divergent selection due to population differences in ecology. Our results do indeed suggest that such counteracting forces might operate, at least in H. impressopunctatus, and possibly also in the other two species of diving beetles in this study. In summary, we have investigated and discussed the selective regimes acting on a polymorphic and sex-limited trait in female diving beetles using indirect inferences (comparisons between neutral markers and morph frequency divergence) and by relating morph frequency divergence to local microclimatic factors, pointing to a role for ecology. In two of the three species, we were not able to reject genetic drift as a main factor driving local population morph frequencies. In the third species, we found statistically significant evidence for divergent selection. These conclusions were further and independently supported by our ecological analyses where we found that local microclimatic factors affected local morph frequencies in a species where drift was rejected but not in the two species where we could not reject drift. The fact that the indirect approach and the approach using environmental factors corroborate each other suggests that our inference about the adaptive causes behind population divergence in this species is robust. The difference in results between these three species might indicate some role for genetic drift affecting population divergence of the morph frequencies, against the effects of local sexual conflict and environmental selection. However, our results do not necessarily exclude a role for stabilizing frequency-dependent selection, because this mode of selection is difficult to detect using the indirect approaches. Instead, the dorsal structures are likely to be influenced by divergent selection caused by local ecological differences, negative frequency dependent selection through sexual conflict and genetic drift, although the relative balance between these different forces are likely to differ between species. More generally, we suggest that the interaction between local ecology

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and sexual conflict dynamics between males and females needs to be considered jointly to understand population divergence in these female traits, just like the interaction between ecology and sexual selection has recently begun to be more appreciated in empirical studies (Gosden and Svensson 2008; Cornwallis and Uller 2010; Karlsson et al. 2010; Svensson and Waller 2013; Miller and Svensson 2014).

ACKNOWLEDGMENTS We are thankful for help from J. Geijer with fieldwork, L. Hathaway with lab work, M. Lundberg for lab work and extracting the AFLP data, and J. Waller for extracting the WorldClim data. F. Rosengren, A. Runemark, M. Wellenreuther, and two anonymous referees provided constructive comments on an earlier draft of this text. This study was funded by the Royal Swedish Academy of Science (a grant to RH) and by the Royal Physiographic Society in Lund (a grant to KKG). The authors declare no conflict of interest.

DATA ARCHIVING The doi for our data is 10.5061/dryad.125gd.

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Associate Editor: A. Chippindale

Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Table S1. Pairwise FST values (for morph locus and neutral variation) obtained with AFLPsurv (Bayesian prior uniform distribution) and Arlequin for H. impressopunctatus with all seven populations included. Table S2. Pairwise FST values (for morph locus and neutral variation) obtained with AFLPsurv (Bayesian prior uniform distribution) and Arlequin for H. impressopunctatus with a subset of five populations included. Table S3. Pairwise FST values (for morph locus and neutral variation) obtained with AFLPsurv (Bayesian prior uniform distribution) and Arlequin for D. lapponicus with all five populations included. Table S4. Pairwise FST values (for morph locus and neutral variation) obtained with AFLPsurv (Bayesian prior uniform distribution) and Arlequin for G. zonatus with all seven populations included. Table S5. Pairwise FST values (for morph locus and neutral variation) obtained with AFLPsurv (Bayesian prior uniform distribution) and Arlequin for G. zonatus with a subset of five populations included. Table S6. Comparisons of FST obtained with Arlequin with FST obtained with AFLPSurv for each analysed group and for both neutral and morph FST. Comparisons are done with pairwise t-tests. Negative FST-values were set to zero. P values are obtained from resampling analyses.

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