High in an old isolated butterfly population

Anniina L. K. Mattila1, Anne Duplouy, Malla Kirjokangas, Rainer Lehtonen, Pasi Rastas, and Ilkka Hanski1

Metapopulation Research Group, Department of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland

Edited by Wyatt W. Anderson, University of Georgia, Athens, GA, and approved July 2, 2012 (received for review April 11, 2012) We investigated inbreeding depression and genetic load in a small reversible way. Theoretical models have been constructed to ana- (Ne ∼ 100) population of the Glanville fritillary butterfly(Melitaea lyze the factors influencing the likelihood of mutational meltdown. cinxia), which has been completely isolated on a small island These models have addressed the effects of effective population [Pikku Tytärsaari (PT)] in the Baltic Sea for at least 75 y. As a refer- size (5) and the rate of appearance of new deleterious ence, we studied conspecific populations from the well-studied and their effects (5), as well as compensatory beneficial mutations metapopulation in the Åland Islands (ÅL), 400 km away. A large (20) and epistatic interactions (21). What is in short supply are population in Saaremaa, Estonia, was used as a reference for es- empirical studies of genetic load and its consequences in natural N timating genetic diversity and e. We investigated 58 traits related populations. Previous studies have rarely documented the history to behavior, development, morphology, reproductive perfor- of isolation and past population sizes. In the absence of this in- mance, and metabolism. The PT population exhibited high genetic formation, researchers have merely assumed that small current load (L=1 − WPT/WÅL) in a range of fitness-related traits including L= fl L population size combined with reduced genetic variation is indic- adult weight ( 0.12), ight metabolic rate ( = 0.53), egg viability ative of long-term isolation and that such populations potentially (L = 0.37), and lifetime production of eggs in an outdoor popula- suffer from the accumulation of deleterious mutations (22). In the tion cage (L = 0.70). These results imply extensive fixation of case of naturally isolated populations, the focus of research deleterious recessive mutations, supported by greatly reduced di- has been on reduced genetic diversity (23–27). Strong support for versity in microsatellite markers and immediate recovery (hetero- fi sis) of egg viability and flight metabolic rate in crosses with other genetic load due to drift is provided by demonstration of tness populations. There was no significant inbreeding depression in recovery (heterosis) in crosses between isolated populations (28, fi most traits due to one generation of full-sib mating. Resting met- 29). Studies of natural populations have demonstrated tness re- abolic rate was significantly elevated in PT males, which may be covery due to a small number of unrelated immigrants entering an – related to their short lifespan (L=0.25). The demographic history isolated population (14, 30 32) and leading to genetic rescue (33, and the effective size of the PT population place it in the part of 34), regardless of whether reduced fitness was due to inbreeding the parameter space in which models predict accumula- depression (segregation load) or drift (fixation load) (35, 36). tion. This population exemplifies the increasingly common situa- Here, we report on inbreeding depression, genetic load (fit- tion in fragmented landscapes, in which small and completely ness), and local adaptation in a population of the Glanville frit- isolated populations are vulnerable to due to high ge- illary butterfly(Melitaea cinxia) on the small island of Pikku netic load. Tytärsaari (PT) in the northern Baltic Sea. This small population has been completely isolated for at least 75 y. As a reference, we fixation load | segregation load | hybrid vigor | persisting population | use the large conspecific metapopulation in the Åland Islands inbreeding avoidance (ÅL), located 400 km away, which has been intensively studied for the past 20 y (37, 38). The amount of neutral genetic variation lost ampant loss and fragmentation of natural habitats is a prime by the small isolated population is consistent with model pre- Rcause of population and species . At their final dictions, given its demographic history. We found no convincing stage, extinctions are typically ascribed to ecological processes evidence for local adaptation and only limited inbreeding de- (1), especially to demographic and environmental stochasticities, pression due to one generation of full-sib mating, supporting which increase the risk of extinction for remaining small pop- a high degree of relatedness between individuals and purging of ulations (2–4). However, in the absence of gene flow, the viability strongly deleterious alleles. In contrast, the isolated PT population of isolated remnant populations may be compromised also by exhibits large fitness reduction in comparison with the reference genetic processes, such as inbreeding depression due to segre- population in a range of traits including body size, flight metabolic gation of deleterious recessive mutations and overdominant rate, egg viability, adult longevity, and lifetime production of eggs alleles and mutational meltdown due to fixation of old and new and larvae. Finally, we observed complete fitness recovery (het- deleterious mutations of smaller effect by random erosis) in crosses between PT and other regional populations, (5–7). On the other hand, increased homozygosity due to in- implying high genetic load in this isolated population, which may breeding and drift may expose strongly deleterious recessive be undergoing a genetic meltdown to extinction. mutations to selection (8), thereby increasing mean fitness (9, 10), and local adaptation may further enhance fitness in the fl – absence of disruptive gene ow (11 13). Complete isolation and Author contributions: A.L.M. and I.H. designed research; A.L.M., A.D., M.K., and R.L. reduction in population size, which are increasingly common performed research; P.R. contributed new reagents/analytic tools; A.L.M., A.D., M.K., predicaments for populations in human-dominated landscapes R.L., and P.R. analyzed data; and A.L.M., A.D., M.K., R.L., P.R., and I.H. wrote the paper. (14–16), can thus have contrasting effects on population viability. The authors declare no conflict of interest. There are scores of studies focusing on the effects of drift or This article is a PNAS Direct Submission. inbreeding or local adaptation (7, 17, 18), but their joint effects Freely available online through the PNAS open access option. remain little studied and therefore poorly understood (19). 1To whom correspondence may be addressed. E-mail: anniina.mattila@helsinki.fi or ilkka. Increasing frequency and fixation of deleterious recessive mu- hanski@helsinki.fi. tations in isolated populations via inbreeding and drift are a par- See Author Summary on page 14744 (volume 109, number 37). ticularly serious concern for conservation as they may erode This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. population viability gradually, in an initially imperceptible but ir- 1073/pnas.1205789109/-/DCSupplemental.

E2496–E2505 | PNAS | Published online August 20, 2012 www.pnas.org/cgi/doi/10.1073/pnas.1205789109 Downloaded by guest on September 29, 2021 PNAS PLUS Results 1+1 founders

Population Origin and Genetic Diversity. The Glanville fritillary 0.8 2+2 founders fi 3+3 founders was rst recorded on the small uninhabited island of PT in the 4+4 founders middle of the Gulf of Finland in 1936. The island has a single 5+5 founders meadow of ca. 10 ha with the larval host plant Veronica spicata 0.7 50+50 founders along the shore. The nearest mainland (Estonia) is ca.30km

away. The Glanville fritillary was most likely unintentionally in- 0.6 troduced from the Estonian coast by people who used the island as a base for fishing and travel. It is practically certain that there 0.5 has been no further gene flow to the island because it has been Heterozygosity visited very seldom by anyone since World War II (WWII) and the butterfly is a poor disperser (39). Exhaustive surveys of the 0.4 entire habitat area on the island revealed 111 and 198 larval family groups (full siblings) in 2009 and 2011, respectively. Using

the formula in Hartl and Clark 2007 (40) and assuming that the 0.3 expected heterozygosities in the initial and current populations 0 20 40 60 80 100 120 are 0.73 and 0.43, respectively (Table 1) and that the age of the Time since colonization population is 100 y (=100 generations), we obtain Ne = 95, Fig. 1. Model-predicted reduction in expected heterozygosity in the PT which is consistent with the above-cited numbers of larval family population with time since the colonization. The results are shown for six groups (some females produce two or even more larval groups different propagule sizes, from a single mated female to 50 females mated that survive until the autumn) (41). with 50 males. Bars giving 1 SD are shown for the propagule size 3+3 and are Data for seven microsatellites indicate closer similarity of the very similar to all other propagule sizes. The propagule is assumed to have PT population to the Estonian population from Saaremaa (SA) the genetic parameters of the Estonian population on the island of Saar- than to the Finnish population from the ÅL (Table 1). The PT emaa (Table 1). The horizontal line gives the expected heterozygosity in the population shares seven alleles in the seven microsatellites with PT population. only the Estonian population, but only one allele with only the < ÅL population (P 0.05). The pairwise Fst values between PT quenced the mitochondrial DNA gene COI, which had only one and Estonia and between PT and ÅL are 0.28 and 0.41, re- haplotype in PT, but this is not informative because the same F spectively, whereas st between Estonia and ÅL is 0.22 (all values haplotype is common in both SA and ÅL, which had five and fi P < statistically signi cant, 0.05). The PT population is geneti- three haplotypes, respectively. cally clearly less diverse than the two other populations (Table The DyadML inbreeding coefficients in Table 1 are nearly 1). The number of alleles in each microsatellite is maximally identical in the three populations, whereas the value of re- four, suggesting that the offspring of a single mated female may latedness is very much higher in PT than in the two other pop- have established the population (taking into account that the ulations (Table 1). These results, and taking into account that SI Mate- introduction most likely took place at the larval stage; the observed (0.47) and expected heterozygosities (0.43) are rials and Methods ). However, modeling the reduction in genetic nearly equal, indicate that there is no excess of nonrandom SI Materials and Methods diversity over time ( ) is more consistent matings in the PT population compared with the other pop- with colonization by the offspring of two or more females (Fig. ulations. Estimates using other models implemented in Coan- 1). The modeling results are consistent with the knowledge that cestry (Materials and Methods) were compatible with those of the population is at least 75 y old, and the results suggest that the DyadML. The overall positive inbreeding values are at least population is unlikely to be older than 100 y (Fig. 1). We se- partly due to null alleles in the microsatellite data.

Life-History Traits and . We measured life-history traits and Table 1. Summary of genetic results for the Pikku Tytärsaari fi (PT, Russia), the Saaremaa (SA, Estonia), and the Åland Islands tness components of individuals mated with full sibs vs. un- (ÅL, Finland) populations related individuals in a common garden laboratory experiment, separately for the PT and ÅL populations (Table 2). Addition- PT SA ÅL ally, we recorded comparable data for other life-history traits, Island area, km2 1.5 2,673 13,517 morphology, and metabolic rates in the laboratory and in a large outdoor population cage (Table 3 and Materials and Methods). Sample size 69 59 28 fl BIOLOGY No. of alleles (SD) 3.57 (0.54) 10.57 (2.44) 8.14 (3.0) For most traits, butter ies from the isolated PT population POPULATION showed significantly lower trait value than butterflies from ÅL Expected heterozygosity (SD) 0.43 (0.09) 0.73 (0.07) 0.72 (0.21) – Observed heterozygosity (SD) 0.47 (0.12) 0.58 (0.14)* 0.53 (0.16)* (Tables 2 and 3; full results in Tables S1 S4). The reduction in † the PT population due to genetic load was measured as Gene diversity (SD) 0.37 (0.22) 0.65 (0.36) 0.62 (0.34) ¼ − = Shared alleles with PT 7 (20) 1 (13) L 1 WPT WÅL, where W PT and WÅL are the average trait only (total) values for outcrossed females in the PT and ÅL populations, respectively. For 9 of the 13 traits L > 0.2, for 4 traits L > 0.4, Fst with PT 0.28 0.41 ‡ Inbreeding DyadML (variance) 0.36 (0.06) 0.31 (0.06) 0.40 (0.05) and for lifetime larval production, the best composite measure ‡ fi Relatedness DyadML (variance) 0.80 (0.10) 0.27 (0.07) 0.39 (0.05) of tness, L = 0.69 (Table 2), implying high genetic load in the PT population. The exceptional trait was the rate of laying egg *All seven markers were included in the analysis although four markers clutches: PT females laid their first egg clutch at a younger age showed indication of null alleles in SA and ÅL populations, probably due than ÅL females, and the average time interval between sub- to a very large size range of alleles. † sequent clutches was shorter in PT females (Table 2). Gene diversity refers to expected heterozygosity over all data and is calcu- The rate of development was lower in the PT population. lated with Arlequin (94). ‡ fi Dyadic maximum-likelihood inbreeding (F; tests for deviation from random Thus, the length of the fth larval instar following diapause was mating) and relatedness values are calculated with the Coancestry (95) pro- ca. 20% longer, and similarly the embryo (Table 2) and pupal gram. Variance refers to the 95% confidence interval for 1,000 bootstrap- development times (Table 3) were about 1 d (ca. 10%) longer in ping samples. the PT than the ÅL population. One exception is the final sev-

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Table 2. Life-history traits and fitness components in outcrossed (between-family) and full-sibling matings in butterflies originating from the old isolated PT population and the large ÅL reference population and the corresponding estimates of inbreeding depression and genetic load ÅL PT PT vs. ÅL§

Inbreeding Inbreeding † ‡ † ‡ Average trait value* t test depression: Average trait value* t test depression: t test Genetic load:

Trait Outcrossed Sib- mated tP δinb Outcrossed Sib- mated tP δinb tP LPT

Prezygotic Timing of reproduction Mating rate, /d, sqrt 1.900 1.650 1.93 0.06 0.13 1.237 1.239 −0.02 0.99 0.00 3.41 0.00 0.35 Oviposition rate, first egg clutch, /d, log 0.343 0.319 1.26 0.22 0.07 0.507 0.539 −0.16 0.87 −0.06 −1.75 0.09 −0.48 Oviposition rate, egg clutches/d, sqrt 0.161 0.138 1.13 0.27 0.14 0.228 0.194 0.89 0.38 0.15 −1.99 0.06 −0.42 Egg clutch size Size of first egg clutch 241 201 1.69 0.10 0.17 148 153 −0.29 0.78 −0.03 4.65 0.00 0.39 Average size of all egg clutches 192 169 1.20 0.24 0.12 118 112 0.34 0.74 0.04 4.75 0.00 0.39 Postzygotic Survival Hatching rate of first egg clutch, %, asin 82 56 2.60 0.01 0.32 64 41 2.40 0.02 0.36 2.36 0.02 0.22 Average hatching rate of egg clutches, %, asin 73 53 2.05 0.05 0.28 46 28 1.85 0.08 0.39 4.81 0.00 0.37 Larval survival before diapause, %, asin 95 73 4.28 0.00 0.23 53 59 −0.21 0.83 −0.11 4.36 0.00 0.44 Development Average embryo development time, /d 0.075 0.073 1.61 0.12 0.02 0.069 0.067 1.85 0.08 0.03 6.90 0.00 0.08 Average larval growth rate, /d 0.045 0.042 3.04 0.01 0.07 0.046 0.048 −0.61 0.55 −0.04 −0.48 0.64 −0.02 Average larval weight, mg 4.5 4.7 −0.56 0.58 −0.04 2.2 2.2 0.38 0.71 0.00 11.84 0.00 0.51 Reproductive fitness Larval group size at diapause, sqrt 190 104 3.38 0.00 0.45 54 43 −0.13 0.90 0.20 5.35 0.00 0.72 Lifetime larval production, sqrt 443 284 1.97 0.06 0.36 139 103 1.30 0.21 0.26 4.13 0.00 0.69

The traits are divided into prezygotic and postzygotic. Transformations of variables: sqrt, square-root transformation; log, logarithmic transformation; asin, arcsine transformation. *The average trait values are calculated from nontransformed data. † Average trait values for the cross types were tested with a t test (using transformed data if a transformation was used). ‡ fi δ ¼ − = Inbreeding depression coef cients inb 1 W sib-mated W outcrossed, where W sib-mated and Woutcrossed are the average values for sib-mated and outcrossed females. § The last three columns give a t test for the difference between the average trait values for outcrossed females from PT and ÅL, WPT and W ÅL , and the genetic load L for PT in comparison with ÅL (LPT ¼ 1 − W PT= W ÅL ). atl tal. et Mattila Table 3. Larval development and other life-history traits and adult morphology and metabolic rate in butterflies PNAS PLUS originating from the old isolated PT population and the large ÅL reference population Female Significance† average‡ Male average‡

Method Pop Sex Pop:sex ÅL PT ÅL PT

Life history and fitness Larval weight at fifth instar, mg CG ** NS NS 5.37 4.55 5.06 4.54 Pupal weight, mg CG **** *** * 187.83 173.78 156.99 150.34 Pupal period, d CG **** NS NS 9.5 10.5 9.4 10.5 Adult longevity, mated, d CG ** * * 23.0 22.3 22.9 17.2 Adult longevity, not mated, d CG **** *** NS 22.9 13.4 17.0 10.3 Probability of flight behavior PC *** ** NS 0.11 0.09 0.23 0.1 Mobility PC NS *** ** −1.33 −0.27 0.95 0.06 Time of day at start of oviposition, h PC **** ——13.42 15.05 —— Lifetime-corrected number of eggs laid PC *** ——52.15 15.41 —— Adult morphology Adult weigt, mg CG **** **** NS 93.8 81.59 55.7 49.68 Thorax weight, age residuals CG **** *** NS 0.52 −0.31 0.69 −0.38 Log abdomen weight, age/mating/ovip residual CG NS ** NS 0.22 −0.23 0.33 −0.18 Wing area, mm2 CG * **** NS 119.33 113.5 97.82 99.03 Wing loading, adult weight/wing area CG *** **** NS 7.77 7.16 5.71 5.05 Aspect ratio, 4 × wing length2/wing area CG NS NS ** 1.13 1.16 1.18 1.13 Outer wing ratio CG *** NS * 0.44 0.427 0.434 0.433 Metabolic rate (weight residual) Pop (F) Pop (M) Pop:Sex

Peak FMR, CO2 mL/h CG, MR **** *** NS 1.68 0.97 1.78 0.95

Peak FMRend,CO2 mL/h CG, MR **** **** NS 0.73 0.32 1.01 0.37 Integrated FMR, CO2 mL CG, MR **** **** NS 0.22 0.11 0.26 0.11

RMR, CO2 mL/h CG, MR NS **** **** 0.11 0.12 0.05 0.1

*P < 0.1; **P < 0.05; ***P < 0.01; ****P < 0.001; CG, common garden experiment; MR, metabolic rate measurement; NS, nonsignificant; ovip, oviposition; PC, population cage experiment; Pop, population. † Shown are the results for models with the fixed effects of population, sex, and their interaction. For metabolic rates (FMR corrected for adult weight, RMR corrected for adult age), the results are shown for models with the fixed effect of population separately for females and males. ‡The last four columns give the average trait values for females and males. The average FMR and RMR values are absolute values, not residuals.

enth larval instar, which was about 1 d (ca. 10%) shorter in fe- number of eggs laid by PT females was about 30% (L = 0.70) of male PT larvae (Table S1). Adult longevity was significantly that in ÅL females (Table 3). Finally, there was an intriguing shorter in PT butterflies, especially in males (25% shorter; Table difference in the timing of oviposition, PT females starting to 3). Among the butterflies that did not succeed to mate in small oviposit on average 1.5 h later in the day than ÅL females (Table cages in the laboratory, most likely because of their inferior 3). This difference was not affected by ambient temperature. condition, lifespan of PT butterflies was about 40% shorter than Lifetime (prediapause) larval production is our best composite the lifespan of ÅL butterflies (both sexes; Table 3). measure of fitness, as it includes several components of female Reproductive performance was very poor in PT males. In the reproductive success, such as the number of egg clutches, egg laboratory mating cages, PT males took significantly longer to clutch size, hatching rate, and larval survival until diapause. On mate than ÅL males (Table 2). In the large outdoor population the basis of this measure, the fitness of the PT population is cage with competition for mates among free-flying males from reduced to 31% of the fitness of the reference population. This

several regional populations (Materials and Methods), only 1 of 18 fitness is, however, likely to be an underestimate, as it does not BIOLOGY POPULATION PT males succeeded to mate, in contrast to 15 of 20 ÅL males that take into account the smaller body mass of PT prediapause mated at least once (χ2 = 22.2, df = 1, P < 0.0001). The low mating larvae (49% of the mass of ÅL larvae) or larval development success of PT males in the outdoor cage was probably partly due to following diapause. their limited flight activity. During regular censuses of butterflies in the cage (Materials and Methods and Table 3, full results in Inbreeding Depression. Inbreeding depression was measured as δ ¼ − = Table S1), PT males were observed to fly with probability 0.10, inb 1 W sib-mated W outcrossed, where Wsib-mated and Woutcrossed compared with 0.23 in ÅL males (χ2 = 6.13, df = 1, P=0.013). are the average trait values for sib-mated and outcrossed The reproductive performance of PT females was inferior to females. The traits are divided into prezygotic and postzygotic that of ÅL females (Tables 2 and 3 and Tables S2 and S4), with traits in Table 2. In the case of prezygotic traits, where the fe- the above-mentioned exception of shorter interval between male is not inbred, there is a trend suggesting higher inbreeding consecutive egg clutches in PT females. This exception is likely to depression in ÅL (average δinb is 0.126 and 0.020 in ÅL and PT, be related to the much smaller average egg clutch size in PT respectively). Mating with a brother reduced the trait values of females (39% smaller; Table 2 and Fig. 2). Consequently, al- ÅL females: They took longer to mate, oviposited at a lower rate, though PT females laid more egg clutches under the common and laid smaller egg clutches than females mated to a nonrelative garden conditions in the laboratory, their lifetime egg production (Table 2 and Fig. 2B). In contrast, there was no such inbreeding was much smaller (∼350 eggs) than that of ÅL females (∼600 depression in PT females in the prezygotic traits (Table 2 and eggs). In the outdoor population cage, the lifetime-corrected Fig. 2A). Among the postzygotic traits, egg viability was signifi-

Mattila et al. PNAS | Published online August 20, 2012 | E2499 Downloaded by guest on September 29, 2021 A PT B PT and ÅL females, namely mating (i) with a full sib, (ii) with an unrelated male from the same population, and (iii) with a male Outcrossed from another regional population (Fig. 3). In the PT population, Sib-mated egg viability was very low in females mated with a sib or a same- population male, but very high in females mated with a male from a different population, indicating very strong heterosis and hence high genetic load. In the ÅL population, sib-mated females had lower egg viability than females mated with an un- related same-population male or with a male from another Eggclutchsize Eggclutchsize population, indicating inbreeding depression.

Adult Morphology. PT larvae were significantly lighter than ÅL 0 50 100 150 200 250 0 50 100 150 200 250 nd nd larvae in all stages of development (Tables 2 and 3), and adult 1st 2 3+rd 1st 2 3+rd PT butterflies were ca. 10% lighter (L = 0.12, average for C D females and males) than ÅL butterflies (Table 3). Apart from the overall size, there were substantial differences in adult mor- phology between the two populations (Table 3, full results in Table S3). Age at dissection affected thorax weight negatively in males (PAGE < 0.001) and females (PAGE < 0.001), whereas in the case of abdomen weight mating had an effect in males (PMATING = 0.02) and age and oviposition event had an effect in females (PAGE = 0.01, POVIPOSITION < 0.001). We corrected for

Hatching rate (%) Hatching rate (%) these effects while comparing the two populations. Both un- corrected and corrected thorax weights were smaller, especially in PT males (Table 3, Tables S3 and S5, and Fig. S1).

0 20406080100 0 20406080100 fl fl 1st 2nd 3+rd 1st 2nd 3+rd Although PT butter ies were smaller than ÅL butter ies, the front wing area was similar in both populations and hence wing Fig. 2. (A–D) Clutch size (A and B) and egg-hatching rate (%) (C and D)in loading was significantly smaller in PT butterflies [wing loading the first two clutches and as the average in the subsequent clutches in PT (A is defined as the ratio of adult weight/wing area, and it is gen- and C) and ÅL females (B and D). Clutches from outcrossed matings (be- erally positively correlated with acceleration capacity but neg- tween family, nPT = 17, nÅL = 20) are shown as shaded bars and clutches from atively correlated with sustained flight (42)] (Table 3 and Table sibling matings (n = 12, n = 20) as open bars. Error bars show the SD. PT ÅL S6, PCA C). Comparing wing area with age-corrected thorax weight, there was a significant population–sex interaction, in- cantly affected by inbreeding in both populations, but prediapause dicating that thorax weight in relation to front wing area was larval survival showed significant inbreeding depression only in more reduced in PT males than females (Table S3). Average the ÅL population (Table 2). Traits related to the rates of embryo front wing length, width, and perimeter and the areas of large and larval development showed no inbreeding depression in ei- and small triangles within the front wing did not differ between ther population, except for larval growth rate, which showed low the populations, but there were subtle significant differences in but significant inbreeding depression in ÅL (Table 2). Unlike the wing shape. PT females had a higher aspect ratio [leaner wings, other traits in Table 2, in which larger values increase lifetime reproductive performance, rates of embryo and prediapause larval development may be under stabilizing selection, with no advan- *** *** ** ** tage from higher rates, and hence these traits do not show in- *** breeding depression (smaller values) in the present analysis. *** Finally, considering larval group size at diapause and lifetime larval production, two compound traits related to lifetime re- productive success, there was high and significant inbreeding de- pression in the ÅL population (the latter was nearly significant with P = 0.06 on the basis of a t test on the square-root trans- formed variable and significant with P = 0.03 on the basis of anonparametricMann–Whitney U test; δinb = 0.45 and 0.36, respectively), but weaker and nonsignificant inbreeding de- Hatching rate (%) pression in the PT population (δinb = 0.20 and 0.26; Table 2). The inbreeding experiment could not be continued after larval diapause (fifth larval instar) due to very low survival of post-

diapause PT larvae. Before the diapause, there were signs of 0 20406080100 0 20406080100

s disease in many groups of PT larvae, and their overall pre- es s ÅL s li ng PT s diapause mortality was much higher (50%) than in ÅL larvae ti tings fami ma crosse crosses b-ma crosse crossesen familie si sib- (5%; Table 2). In terms of lifetime production of diapause larvae we the fitness of PT females was only 31% of the fitness of ÅL between bet females (Table 2), but in reality the difference was much greater as the PT larvae were only half of the size of the ÅL larvae Fig. 3. Egg-hatching rate (%) in three types of matings in ÅL and PT pop- ulations. The mating types are crosses between five regional populations, (Table 2) and apparently in poor condition. including ÅL and PT (Materials and Methods; n = 11, n = 14 egg clutches), In the course of the outdoor cage experiment, we obtained PT ÅL crosses between different families from the same regional population (nPT = naturally occurring crosses within and between PT and four 57, nÅL = 85 egg clutches), and sib mating (nPT = 60, nÅL = 58 egg clutches). other regional populations (Materials and Methods). In the case The lines above the graphs indicate significant differences between pairs of of egg viability, we compared three types of matings for both treatments: **P < 0.01; ***P < 0.001.

E2500 | www.pnas.org/cgi/doi/10.1073/pnas.1205789109 Mattila et al. Downloaded by guest on September 29, 2021 generally associated with fast flight (42)], whereas in males the 0.001), and the hybrid males had similar RMR to that of ÅL PNAS PLUS aspect ratio was lower in PT butterflies (significant population– males (Fig. 5). sex interaction, Table 3). In a principal component analysis (Table S6, PCA D), the component that is analogous to aspect Discussion ratio differed significantly between the populations in both When an isolated population remains small for an extended females and males. period, some of the mutations present at the time of isolation become fixed, whereas others become purged (5, 9, 43). At the Flight and Resting Metabolic Rates. The flight metabolic rate same time, new detrimental mutations enter the population, and (FMR) was positively correlated with adult weight in both the smaller their effect the higher is the probability of their fix- fl sexes, whereas butter y age had a slight negative effect on ation (44, 45). Given enough time, such a population is likely to resting metabolic rate (RMR). While comparing the two show reduced fitness due to high load of fixed mutations, but no populations below, we used residuals from linear regressions reduced fitness upon further inbreeding. The reduction in in- accounting for these effects. breeding depression is due to increased homozygosity: Fixed loci The FMR of PT butterflies was only half of that of ÅL but- fl show no inbreeding depression, whereas a part of the mutations ter ies (average genetic load of FMR measures L = 0.53; Table with the strongest deleterious effects have been purged (35, 46, 3 and Figs. 4 and 5). The difference was evident in both peak and 47). The old isolated population of the Glanville fritillary on the integrated FMR, but was especially notable at the end of the 15- island of PT in the Baltic Sea represents a prime example. First, min flight period (Fig. 4 and Table 3), when the PT butterflies the fitness of the PT population is reduced to <30% of the fitness became exhausted sooner and more severely than ÅL butterflies. of the large reference population in the ÅL (on the basis of The ÅL males typically exhibited multiple peaks of CO emission 2 lifetime larval production, our best composite measure of fitness, during the 15-min experiment, but in PT males CO emission 2 and the expected performance of the larvae following diapause), peaked only in the beginning of the experiment. A principal fi component analysis supports these conclusions (Table S6, PCA representing one of the greatest tness declines reported for B). There was no difference in the RMR between the pop- natural populations. Second, the PT population has much less ulations in females, but in males the difference was highly sig- genetic variation in microsatellite loci than the ÅL population, fi nificant and the reversed of that in FMR, PT males showing consistent with large-scale xation of alleles by genetic drift. almost twice as high RMR than ÅL males (Fig. 5). The ratio Third, in the PT population most traits were little or not at all FMR/RMR (a measure of metabolic scope) was much higher in affected by one generation of full-sib mating, in contrast to δ ∼ ÅL females (15.3) and especially males (35.6) than in PT females substantial inbreeding depression ( inb 0.25) in the ÅL pop- (8.1) and males (9.5). ulation. Fourth, hybrid offspring between the PT butterflies and Between-population PT crosses showed strong heterosis in individuals from other regional populations showed complete metabolic rates (Fig. 5). The offspring of PT females crossed fitness recovery in the two traits studied, egg viability and flight with a male from another regional population [ÅL, Saaremaa, metabolic rate. Finally, we found no clear evidence for local Uppland (Sweden), or Öland (Sweden)] had significantly higher adaptation, which is discussed further in Female Performance. peak FMR than pure-bred PT butterflies [Tukey’s honestly sig- Reduced genetic variation in the isolated population is likely to nificant difference (HSD), P < 0.001 for both males and constrain local adaptation (48). females], which was not significantly different from the FMR of Examples of fitness reduction in small isolated populations ÅL butterflies. The significantly elevated RMR in PT males include studies on mammals [Weddell seals (16)], reptiles and (Table 3) entirely disappeared in the crosses (Tukey’s HSD, P < amphibians [adders (15, 49), timber rattlesnakes (50), European BIOLOGY POPULATION CO2emissionrate(mlCO2/h) CO2emissionrate(mlCO2/h) 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5

0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200

Time (sec) Time (sec)

Fig. 4. The CO2 emission curves during the 15-min flight experiment (including the base line) for five randomly selected females and males from the two populations. Butterflies from PT are represented by thick solid lines and butterflies from ÅL by thin dashed lines.

Mattila et al. PNAS | Published online August 20, 2012 | E2501 Downloaded by guest on September 29, 2021 of deleterious mutations present in the population at the time of establishment and to what extent it is due to fixation of newly arisen mutations, although given the relatively short time since colonization, it is likely that the majority of fixed deleterious mutations arise from standing detrimental variation.

Female Performance. In the case of female reproductive perfor- mance, both fecundity (lifetime egg production) and offspring viability were significantly reduced in the PT population in com-

PT PTxXX ÅL ÅLxÅL PT PTxXX ÅL ÅLxÅL parison with the reference ÅL population. There was limited in- breeding depression in the PT population, with one notable Population/cross Population/cross exception: Inbreeding depression in egg viability was even greater in the PT (δinb = 0.39) than in the ÅL population (δinb = 0.28; average for all clutches, Table 2), although there was also a sig- nificant reduction in egg viability in outcrossed females in the PT compared with the ÅL population (L = 0.37), indicating fixation load. Mutations affecting egg viability operate early in the life cycle, and often such mutations are more deleterious than muta- tions expressed later in the life cycle (62). As purging of highly deleterious mutations is more efficient than purging of mildly deleterious mutations, other things being equal, the high in-

PT PTxXX ÅL ÅLxÅL PT PTxXX ÅL ÅLxÅL breeding depression of egg viability in the PT population is sur- prising. Other studies have previously reported that purging may Population/cross Population/cross not effectively reduce inbreeding depression in all traits (63, 64). Fig. 5. Peak flight metabolic rate (Upper, residual from a linear model in- Noninbred females in the ÅL reference population took longer cluding adult weight) in females and males and resting metabolic rate (Lower, to mate and oviposit and laid smaller egg clutches, when mated to residual from a linear model including butterfly age at measurement) in a brother as opposed to an unrelated male. In contrast, there was females and males in four lines of butterflies: PT, butterflies reared from field- no such effect in PT females. The parsimonious explanation of × collected PT larvae (shaded boxes, nfemales =25,nmales =25);PT XX, crosses these results is kin recognition and inbreeding avoidance in the between PT females and males from other regional populations (boxes with shaded hatched lines, n =16,n = 18); ÅL, butterflies reared from ÅL population but not in the PT population, in which all indi- females males viduals are highly related. One previous study found no evidence field-collected ÅL larvae (open boxes, nfemales =25,nmales =25);andÅL×ÅL, crosses between different families from the Åland metapopulation (open for kin recognition in the Glanville fritillary (65) but it used dif-

boxes, nfemales =9,nmales =12).ThePT×XX and ÅL×ÅL crosses were obtained ferent methods from those of the present study. from the outdoor population cage (Materials and Methods). We did not find any clear evidence for local adaptation in the PT population, although this result does not rule out the possi- bility of local adaptation in traits not investigated in the present tree frogs (28, 51), and natterjack toads (52)], fish [Japanese rosy study. Here, we define local adaptation as a trait that is expected bitterling (53)], aquatic snails (54), and plants [Carex davalliana to increase, on the basis of biological knowledge, the fitness of its (55)]. In these studies, the fitness of the isolated population carrier. An adaptive trait may be specific to a certain environ- ranged from 30% to 90% of the fitness of the corresponding ment (e.g., low dispersal rate in an isolated island population) or nonisolated conspecific population, with an average of 65%, al- a trait may be adaptive more generally (e.g., high fecundity). though it should be noted that the fitness components and the There are two female traits in the PT population that at first methods used varied greatly between the studies. These studies could be interpreted as indicating local adaptation: reduced examined on average 3.5 fitness components (range from 2 to 6), FMR and high rate of laying egg clutches. In the Glanville frit- whereas our study has reported 58 traits (Table S7) for natural illary, FMR and dispersal rate in the field are strongly correlated populations of the Glanville fritillary. Not all traits studied here (66), and hence reduced FMR in PT could be interpreted as are independent of each other, for which reason we summarized being an adaptation to an isolated island, where high dispersal variation in morphometric and metabolic traits with principal rate is most likely selected against (67, 68). However, the local component analyses. Studying only a part of the life cycle is likely adaptation hypothesis is not supported by the observation that to lead to an underestimate of the true fitness effect (56, 57). In high FMR has contrasting effects on dispersal propensity in the some studies, for instance in the case of the Florida panther (32, two sexes; only females with high FMR are more likely to dis- 58, 59) and the greater prairie chicken (14), researchers have perse, whereas males with high FMR are actually less dispersive demonstrated fitness recovery via genetic rescue when individuals than males with low FMR, but the former benefit from high from other populations were introduced to isolated populations. FMR through their superior capacity to acquire mates in the Models predict that mutation accumulation may generate a natal population (69). In the PT population, FMR was similarly significant risk of extinction in small populations (5, 44). Lynch reduced in both sexes. Second, in crosses between PT and other et al. (5) concluded that sexual populations with an effective size populations FMR was at the level of that of the reference ÅL smaller than 100 individuals are unlikely to persist for more than butterflies, which is most parsimoniously explained by heterosis. a few hundred generations, assuming mutation parameters based Third, flight morphology of PT females does not indicate re- on Drosophila studies. The theory and empirical results on mu- duced flight ability. PT females had a smaller thorax than ÅL tational meltdown are, however, controversial (60), and the females, but they had a higher wing aspect ratio [associated with impacts depend on the nature of the genetic load (61). Mutational fast flight (42)] and smaller wing loading [generally associated meltdown is more likely to take place with a predominance of with low acceleration capacity but higher capacity for sustained mutations of small effect (35). Our results are in agreement with flight (42)] than ÅL females. We therefore suggest that low FMR the prediction of mutational meltdown within an order of 100 in PT butterflies is another consequence of large genetic load generations in populations with an effective size of 100 or less. It (see Male Performance for resting metabolic rate in males). remains an open question to what extent the high genetic load in Turning to the larger number of smaller egg clutches laid by our study population is due to fixation (or drift to high frequency) PT than by ÅL females, this difference could possibly be an ad-

E2502 | www.pnas.org/cgi/doi/10.1073/pnas.1205789109 Mattila et al. Downloaded by guest on September 29, 2021 aptation to spread the risk of entire clutches being parasitized by ulations are vulnerable to extinction due to fitness decline caused PNAS PLUS the specialist parasitoid Cotesia melitaearum, which occurs on PT by high genetic load. (Materials and Methods), especially because parasitism is known to increase with the size of larval groups (70). The average rate Materials and Methods of parasitism must be higher on PT than in ÅL, because most Study Species and Populations. PT is a small (1 × 2 km) and very isolated small local populations in ÅL lack the parasitoid (71). On the Russian island in the Gulf of Finland, located ∼30 km north of the Estonian other hand, apart from the effect of parasitism by Cotesia, larval coast. The island has a single shoreline meadow of about 10 ha with V. survival increases with group size (72), which should select for spicata, the larval host plant of the Glanville fritillary. The Glanville fritillary fl was recorded on the island in 1936 and again in 1994, and it has persisted larger groups. Another hypothesis is that PT butter ies have until the present. The island has been uninhabited by people, but it was evolved a faster adult life history, but it is not clear why that would frequently visited before WWII by fishermen coming from the nearby larger be especially advantageous in their environment, and in fact it is island of Tytärsaari. Material has been brought to the island from the Es- not, as the lifetime number of eggs produced by PT females was tonian coast, most likely leading to accidental introduction of larvae. In- only 30% of that produced by ÅL females. Finally, a nonadaptive troduction at the larval stage is supported by the presence of the specialist explanation of the oviposition pattern is that PT females are braconid parasitoid C. melitaearum, which is an even worse disperser than constrained to lay small egg clutches, which take a shorter time to the butterfly (71) but could have arrived as parasitized host larvae. The PT mature than large clutches, and hence PT females are able to lay population was surveyed thoroughly in late August 2009, and 111 larval family groups were detected. Five larvae were sampled per group. For the next egg clutch sooner than ÅL females. comparison, we sampled three larvae per larval family group from ca. 100 fi different local populations in the well-studied large metapopulation in the Male Performance. In the case of males, tness reduction in the ÅL in Finland (38, 90). The total census size of the ÅL metapopulation is some PT population is dramatic and leaves little scope for alternative tens of thousands of individuals (38), but the effective metapopulation size interpretations. At the physiological level, the flight metabolic is much smaller, probably of the order of 1,000 individuals. Postdiapause rate was greatly reduced, as in females. Additionally, the RMR larvae were reared in 2 groups, taken out of diapause either in February of PT males was twice the rate of ÅL males. Increased RMR may (group 1) or in April 2010 (group 2). Details of the rearing conditions are increase the oxidative stress that is associated with elevated en- given in SI Materials and Methods. After eclosion, butterflies were sexed and marked individually with a number on the underside of the hind wing. ergy use. Males in the PT population had an especially short fl fl lifespan (Table 3), which can be related to their high RMR (73, Butter ies from group 1 were assigned to either measurement of ight and resting metabolic rates or an inbreeding experiment. Group 2 butterflies 74), although we realize that association between RMR and were released 24 h after eclosion into a large outdoor population cage. lifespan is still under debate (75). Studies examining the effect of increased homozygosity (inbreeding) on metabolic rate are Microsatellite Genotyping and Analyses. DNA was extracted with the Nucleo- scarce (76–78). A study on the cricket Gryllodes sigillatus (only Mag 96 Tissue system (Macherey-Nagel) according to the manufacturer’s males) found an 80% increase in RMR of inbred crickets over protocol from 156 unrelated butterflies from the PT and ÅL populations and that in outcrossed individuals, along with a slight decrease in a population from the large island Saaremaa in Estonia. Samples were metabolic rate during forced exercise (79). Elevated levels of genotyped for seven microsatellites, including AM0, AM1, AM2, AM3, and oxidative stress have been reported in inbred Drosophila males AM4 described by Sarhan (91) and two new ones selected from the genome (80), and other studies have shown reduced lifespan in pop- assembly of the Glanville fritillary (details in SI Materials and Methods). Design of robust microsatellite primers is difficult for (92), and ulations suffering from inbreeding or genetic load (81, 82). the microsatellites have often null alleles, due to loss of the longer allele Reduced mass-specific FMR combined with reduced mass of (93). The allele distributions and Fst values were calculated with Arlequin (94) flight muscles go some way toward explaining the reduced flight and the within-population dyadic maximum-likelihood inbreeding (F)and activity of PT males in the population cage. Reduced flight ac- relatedness values (denoted as DyadML estimates) with Coancestry (95). The tivity, in turn, may explain at least partly the reduced mating nine-parameter model of inbreeding based on within-population pairwise success of PT males while in competition with males from other identity-by-descent (IBD) estimates (96) was used as originally implemented populations. Previous studies on have demonstrated that by Wang (97). The DyadML relatedness method can be used in the presence fl of inbreeding and genotyping errors (96). In Table 1, “gene diversity” refers reduced ight activity of males decreases their mating success (83, “ 84). The dramatically reduced egg-hatching rate of the second to the expected heterozygosity over all data, whereas the expected het- erozygosity” is the mean for individual loci [see Arlequin 3.5 documentation, and subsequent egg clutches in the PT population is probably section 8.1. (94)]. another indication of low performance of PT males, which may transfer small amounts of or low-quality sperm during matings. In Decline in Heterozygosity Since the Colonization. We conducted simulations to Drosophila simulans, males suffering from inbreeding depression estimate the number of founding individuals of the PT population, using transfer smaller amounts of sperm (80), whereas in the butterfly a simulation software similar to that in Gasbarra et al. (98). The simulation is

done in two phases. In the first phase, a random pedigree is constructed BIOLOGY

Bicyclus anynana, egg-hatching rate is more affected by the in- POPULATION breeding of the male than of the female (85). The general adverse backward in time, from the youngest generation toward the founders. In effect of inbreeding on male reproductive performance in insects the second phase, neutral genetic data for each individual in the pedigree has been reported for Drosophila melanogaster (80, 86, 87), the are simulated forward in time. Generations are assumed to be non- fl overlapping, consistent with the biology of the Glanville fritillary. The sim- butter y B. anynana (88), and the Glanville fritillary (89). ulation was repeated 100 times and the expected heterozygosity over the seven markers was computed for generations t = 0, 10, ... , 120. The mean Conclusion values for different propagule sizes were compared with the expected Our results on the Glanville fritillary indicate a high load of fixed heterozygosities for the PT sample (Fig. 1 and Table 1). For details see SI or near-fixed deleterious recessive mutations in an old isolated Materials and Methods. population. The demographic history and the size of the pop- ulation place it in the part of the parameter space where models Larval Development and Adult Morphology. Postdiapause larval development predict the possibility of mutational meltdown. Given the rela- was studied using larvae from group 2. The dates of the larval molts and their tively short time since colonization, it is likely that the majority of weights in the beginning of each molt were recorded individually (Mettler- fixed deleterious mutations originate from standing detrimental Toledo XS 105 analytical balance, accuracy 0.01 mg). Pupae were weighed on the day after pupation and the length of the pupal period was recorded. variation, whereas the role of newly arisen mutations would in- Adult and abdomen weights were measured for 51 PT and 50 ÅL butterflies, crease with time. Our results suggest that the PT population adult weight immediately following the measurement of flight metabolic exemplifies the increasingly common situation in human-frag- rate (below). Thorax weight was measured for all PT adults (n = 176) and for mented landscapes in which small and completely isolated pop- most ÅL butterflies (n = 106). For details see SI Materials and Methods.

Mattila et al. PNAS | Published online August 20, 2012 | E2503 Downloaded by guest on September 29, 2021 Wings were detached from the thorax, using tweezers (n = 51 and n =50 males). We could not obtain any PT × PT offspring for these measurements. for PT and ÅL, respectively). One forewing was chosen to represent each The flight metabolic rate was measured using flow-through respirometry in individual. The forewings were photographed against a white background, the constant temperature of 30 °C [see SI Materials and Methods and Nii-

using a digital camera (Canon PowerShot A710 IS) and a ruler for scale. The tepõld et al. (66) for details]. We used the average CO2 emission rate during image analysis software ImageJ 1.37v (Wayne Rasband, http://rsb.info.nih. 60 s of stable baseline before the 15-min flight experiment as a measure of

gov/ij/) was used for wing measurements (accuracy 0.01 mm). Forewing RMR. Peak FMR refers to the maximum rate of CO2 emission during the length, width, perimeter, area, areas within the small and large triangles flight, integrated FMR is the total volume of CO2 produced during the 15- within the wing, wing loading, and aspect ratio were measured as described min experiment, and peak FMREND and integrated FMREND represent the in SI Materials and Methods. peak CO2 emission and the total CO2 volume produced during the last 5 min of the experiment, respectively. Measures of FMR and RMR were corrected Population Cage Experiment. The large outdoor population cage (32 × 26 × for variation in body mass and age, respectively, by using residuals from 3 m) covered a dry meadow rich in flowering nectar plants and gardened linear regressions. to resemble the natural habitat of the Glanville fritillary (99, 100). The ex- perimental setup was the same as used by Saastamoinen and Hanski (99). Statistical Analyses. Statistical analyses related to the inbreeding experiment, Altogether 207 butterflies (group 2) were released into the cage in June 2010, the population cage experiment, and adult morphology and metabolism were representing the following regional populations: 13 females and 18 males carried out using PASW Statistics 17.0, R Version 64, and R Version 2.10.1 (101), from PT, 14 and 21 from ÅL, 31 and 27 from Saaremaa, 18 and 26 from respectively. We used two-way ANOVA to test for the effects of the pop- Uppland, and 17 and 22 from Öland. Matings and ovipositions occurred nat- ulation of origin (PT or ÅL) and sex on the morphological, physiological, fl fl urally by free- ying butter ies and were constantly monitored, along with developmental, life-history, and fitness-related traits (details in SI Materials fl regular censuses of butter ies in the cage (SI Materials and Methods). Fol- and Methods). Resting metabolic rate and peak flight metabolic rate were lowing oviposition, the leaf supporting the egg clutch was collected and the compared in the first (field-collected)- and second (laboratory-reared)-gen- eggs were reared in the laboratory (SI Materials and Methods). The eggs were eration butterflies (PT × “another population” and ÅL × ÅL crosses, as counted at the age of 3 d. Following egg hatching, the larvae were counted. explained above), using ANOVA and Tukey’s HSD tests. Many of the traits that were included in this study are correlated, and we therefore performed Inbreeding Experiment. An inbreeding experiment was conducted with but- several principal component analyses on traits related to metabolism and fl ter ies from the PT and ÅL populations (group 1) by comparing within- morphology. The methods are given in SI Materials and Methods and results family crosses (full-sib mating, nPT = 12, nÅL = 20) with between-family in Tables S5 and S6. For traits included in the inbreeding experiment, the fl crosses (nPT = 17, nÅL = 20). Butter ies were mated in small cylindrical net difference between average trait values of between-family vs. within-family × cages (13 30 cm) in the laboratory. After mating, the females were isolated crosses was tested with a t test. Oviposition rate of the first egg clutch was log and allowed to oviposit on individual potted host plants (SI Materials and transformed; egg hatching rate and larval survival rate were arcsine trans- Methods). Oviposition rate refers to the number of egg clutches laid per day formed; and oviposition rate, larval production, larval group size, and mating during the lifetime of a female, including the females that laid no egg rate were square-root transformed to make the variables normally distrib- clutches. The numbers of eggs and larvae per group were counted. For each uted. Nonparametric Mann–Whitney U tests gave qualitatively similar results. female, the first larval group was reared until diapause (SI Materials and Methods). The average larval weight at diapause was measured, and de- ACKNOWLEDGMENTS. We thank Elena Glazkova for surveying and sam- velopment time and mortality of larvae were recorded. Adult longevity was pling the PT population; Suvi Ikonen, Coong Lo, Elli Lappalainen, Annukka fl recorded separately for butter ies that mated in the experiment, those that Ruokolainen, and Toshka Nyman for technical assistance; Christoph Haag, did not succeed to mate, and those that did not participate in the mating Ilik Saccheri, and two anonymous referees for comments on the manuscript; experiment (no opportunity to mate). Kristjan Niitepõld for advice on insect respirometry; Marjo Saastamoinen and Maaike De Jong for discussions; the many research assistants who took Flight and Resting Metabolic Rates. FMR and RMR were measured for 25 part in the population cage experiment and helped to collect and rear the larvae; and the staff of the Lammi Biological Station. A.L.M. was funded by females and 25 males from both populations in 2010. Each individual was the Finnish School in Wildlife Biology, Conservation, and Management. This chosen from a different family. In the following generation, we measured the study was supported by European Research Council Grant AdG 232826 (to metabolic rates of the offspring of PT females mated with males from an- I.H.), the Academy of Finland (Finnish Center of Excellence Programme other regional population (ÅL, Saaremaa, Uppland, or Öland) (16 females Grants 131155, 38604, and 44887; to I.H.), and the Lammi Biological Station and 18 males) and of the offspring of ÅL × ÅL crosses (9 females and 12 Foundation of Environmental Research (M.K.).

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