Dynamics of Genetic Rescue in Inbred Drosophila Melanogaster Populations

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Springer - Publisher Connector Conserv Genet (2010) 11:449–462 DOI 10.1007/s10592-010-0058-z

RESEARCH ARTICLE

Dynamics of genetic rescue in inbred Drosophila melanogaster populations

R. Bijlsma • M. D. D. Westerhof • L. P. Roekx • I. Pen

Received: 11 October 2009 / Accepted: 23 December 2009 / Published online: 4 February 2010 Ó The Author(s) 2010. This article is published with open access at Springerlink.com

Abstract Genetic rescue has been proposed as a man- deleterious at 25°C but lethal at 29°C. By comparing fitness agement strategy to improve the fitness of genetically eroded at 25°C (the temperature during the rescue experiment) and populations by alleviating inbreeding depression. We stud- 29°C, we show that the lethal allele reached significant ied the dynamics of genetic rescue in inbred populations of frequencies in most rescued populations, which upon Drosophila. Using balancer chromosomes, we show that the renewed inbreeding became fixed in part of the inbred lines. force of heterosis that accompanies genetic rescue is large In conclusion, in addition to the fitness increase genetic and allows even a recessive lethal to increase substantially in rescue can easily result in a substantial increase in the fre- frequency in the rescued populations, particularly at stress quency of mildly deleterious alleles carried by the immi- temperatures. This indicates that deleterious alleles present grants. This can endanger the rescued population greatly in the immigrants can increase significantly in frequency in when it undergoes recurrent inbreeding. However, using a the recipient population when they are in linkage disequi- sufficient number of immigrants and to accompany the librium with genes responsible for the heterosis. In a second rescue event with the right demographic measures will experiment we rescued eight inbred Drosophila populations overcome this problem. As such, genetic rescue still is a with immigrants from two other inbred populations and viable option to manage genetically eroded populations. observe: (i) there is a significant increase in viability both 5 and 10 generations after the rescue event, showing that the Keywords Drosophila Á Gene flow Á Genetic drift Á increase in fitness is not transient but persists long-term. (ii) Genetic load Á Genetic rescue Á Inbreeding Á The lower the fitness of the recipient population the larger Inbreeding depression the fitness increase. (iii) The increase in fitness depends significantly on the origin of the rescuers. The immigrants used were fixed for a conditional lethal that was mildly Introduction

Human impact on nature has caused habitats to become R. Bijlsma (&) Á M. D. D. Westerhof Á L. P. Roekx increasingly fragmented. As a result, populations of many Population and Conservation Genetics, Centre for Ecological species have become small and greatly isolated (Ceballos and Evolutionary Studies, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands and Ehrlich 2002). Typically, such populations are subject e-mail: [email protected] to genetic drift and inbreeding with associated decline in fitness due to inbreeding depression (Frankham 1995; R. Bijlsma Á I. Pen Hedrick and Kalinowski 2000). This fitness decrease is for Theoretical Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, P.O. Box 14, 9750 AA Haren, the greater part thought to be caused by fixation of recessive The Netherlands detrimental variation (Hedrick 1994; Charlesworth and Charlesworth 1999; Wang et al. 1999) thereby significantly R. Bijlsma elevating the extinction risk of populations (Newman and Department of Biological Sciences, Ecology and Genetics, Aarhus University, Ny Munkegade, Buildg. 1540, Pilson 1997; Saccheri et al. 1998; Bijlsma et al. 2000). 8000 Aarhus C, Denmark Although the importance of inbreeding depression in 123 450 Conserv Genet (2010) 11:449–462 conservation biology has been hotly debated (Caughley immigrants and linked to the genes that are responsible for 1994; Hedrick et al. 1996), there is now increasing evidence the heterosis may reach appreciable frequencies in the res- that inbreeding depression is present in population of many cued populations. In later generations this may cause addi- wild living species (Keller and Waller 2002) and does tional fitness problems, particularly when Ne is not greatly significantly contribute to the extinction risk of populations increased by the rescue event. In the first experiment we (Hedrick 1995; Spielman et al. 2004; Liberg et al. 2005). explore this possibility by ‘‘rescuing’’ ten independently To prevent populations to become extinct from inbreed- inbred populations of D. melanogaster by immigrants that ing, inbred populations may be genetically rescued and have carry chromosome balancers that are lethal in homozygous their fitness partly restored by immigration of a few unre- condition. We show that in some cases such chromosome lated individuals from another population (Tallmon et al. balancers do significantly increase in frequency despite the 2004; Hedrick 2005; Edmands 2007). This is for the greater fact that these chromosomes carry a recessive lethal. part due to the fact that the infusion of unrelated genomes To test if genetic rescue leads to a more long-term fitness increases heterozygosity in the recipient population, thereby increase, we rescue inbred populations with immigrants reducing the inbreeding load caused by the fixation of from either one of two other inbred populations that carry a recessive deleterious alleles, though other processes can conditional recessive lethal system that is not expressed at a play a significant role (e.g. frequency dependent selection benign temperature but causes high pre-adult mortality for rare S-alleles in plants (Leducq et al. 2010)). This con- under high temperature stress. We assess the increase in cept of genetic rescue is now considered to be a realistic viability (the rescue effect) compared to the non-rescued management option to counteract the increased extinction populations at two different time points, five and ten gen- risk from genetic erosion (for reviews see Tallmon et al. erations after the rescue event. We show that a positive 2004; Hedrick 2005; Edmands 2007), and has been shown to rescue effect is still observed after ten generations but that be very effective in a number of cases (Hedrick 1995; We- the magnitude varies considerably between both the dif- stemeier et al. 1998; Madsen et al. 1999; Willi et al. 2007; ferent rescued populations and the assay temperatures. Bouzat et al. 2009; Hedrick and Fredrickson 2010). Applying genetic rescue as a management measure is However, this process is not without potential dangers primarily meant to increase the fitness of the rescued (see Tallmon et al. 2004; Edmands 2007; Hedrick and population and to increase the population numbers in the Fredrickson 2010). First, immigration by genetically diver- long-term. Therefore, genetic measures have necessarily to gent individuals can disrupt both intrinsic coadaptation and be complemented by demographic and environmental local adaptation (Hedrick 1995; Edmands 2007) and result in measures (Robert et al. 2007). However, given the current outbreeding depression (Dobzhansky and Pavlovsky 1958). level of habitat fragmentation, the population size probably To what extent these potential fitness costs of genetic rescue may increase to a certain extent, but still will be (very) are larger than the fitness benefits is, therefore, an important limited. In these situations recurrent inbreeding cannot be issue in conservation genetics. Second, when the immigrants prevented and the resulting genetic deterioration may are successful their genomes can increase rapidly and dis- become as bad or even worse than before. This has been proportional in frequency in the recipient population in a few observed in an isolated population of gray wolves in generations (Ball et al. 2000; Ebert et al. 2002; Saccheri and Scandinavia that was initially rescued by a single migrant Brakefield 2002). As such, the immigrant genomes become where after the population numbers increased (Vila` et al. overrepresented in the rescued population thereby reducing 2002). More recently, however, this population has been the genetically effective population size, Ne, rather than shown again to be severely inbred and its persistence is increasing it (Hedrick and Fredrickson 2010). Third, the fit- greatly endangered due to inbreeding depression (Liberg ness benefits may be short-lived as recombination may dilute et al. 2005). To investigate the consequences of recurrent the heterotic effect in subsequent generations (Lynch 1991). inbreeding we subject rescued populations to renewed The aim of this paper is to explore some of the potential inbreeding and test the fitness of the resulting inbred lines dangers of genetic rescue using Drosophila melanogaster as again at 25 and 29°C. We observe that the introgressed a model organism. Based on the assumption that heterosis conditional lethal can become prevailing in this situation. from covering up the expressed load from deleterious mutations in the rescued population leads to a rapid spread of the immigrant genomes (Ball et al. 2000; Saccheri and Materials and methods Brakefield 2002), we hypothesize that this not only holds for the loci that are under positive selection but also for genes in Flies and inbreeding procedure linkage disequilibrium with these loci (Maynard Smith and Haigh 1974). Moreover, if the heterotic selection pressure is The inbred flies used for the experiments originated from strong enough even rare deleterious alleles present in the the Groningen’83 base population. This population was 123 Conserv Genet (2010) 11:449–462 451 founded by 403 inseminated females caught at a local fruit females males market in Groningen (The Netherlands) in 1983 and kept as CyO TM3 a large population since then (see Vermeulen and Bijlsma X 2006). Previous experiments have shown that this popula- Pm Sb tion still exhibits substantial levels of genetic variation and still harbours a considerable load (Bijlsma et al. 1999; CyO TM3 Vermeulen and Bijlsma 2004a, b). Flies were normally X maintained on standard medium (1,000 ml water, 32 g dead yeast, 54 g sucrose, 18 g agar and 13 ml nipagin solution (10 g nipagin in 100 ml ethanol)) at 25°C, 50% R.H. and constant light. CyO TM3 Inbred lines were established from the base population through four consecutive generations of full-sib mating resulting in a theoretically inbreeding level of f = 0.594 (Falconer and Mackay 1996). To this end, single pairs of flies were randomly established in vials and from the off- CyO TM3 spring of each pair five full-sib pairs were established 10 individually in vials. For the next round of inbreeding X 50 again five single pairs were established from that of the five vials of the previous generation that on visual inspection 40 produced the most offspring. This procedure selects for high productivity and allows for within line purging of highly deleterious alleles (Wang 2000). This mimics in part Fig. 1 Crossing scheme to produce females that are heterozygous for the process of purging as it would occur in small popula- the balancer chromosomes CyO and TM3 in an inbred background tions and avoids that ending up with greatly crippled lines (top). ?i Denotes chromosomes originating from the inbred population i. Note that this scheme ensures that the X-chromosomes are all that would never occur under natural conditions. When originating from the inbred population. The bottom part (within the reaching the desired level of inbreeding, the lines were oval) shows the initial composition of the rescued populations expanded and maintained as bottle populations (300–400 individuals per generation) for several generations prior to the experiments. Of these lines the first ten remaining very small 4th chromosome) for each of the inbred popu- (I(nbred)1, I3, I5, I6, I7, I8, I11, I12, I15 and I17) were lations separately. The X-chromosomes all originate from used for the experiments described here. Throughout the the inbred population. These females were used as paper, we will refer to these as inbred populations. migrants and for each inbred population 10 of these females, virgins, were combined with 40 virgin females and 50 males from the inbred population and used as initial Balancer chromosome introgression experiment parents for the rescued populations (Fig. 1). This results in an initial frequency of each of the balancers of 5% in each To test whether or not deleterious alleles can invade in an population. The next generation, all offspring were trans- inbred population we used the 2nd chromosome balancer ferred to a new bottle to serve as parents for the next CyO and the 3rd chromosome balancer TM3. Chromo- generation. After a sufficient number of eggs was produced somes 2 and 3 are marked with visible (dominant) muta- (200–500 eggs per bottle) these flies were removed and tions curled wings (Cy) and serrate wings (Ser), phenotypically scored for the presence (heterozygotes) or respectively (for description of balancers and markers see absence (wildtype inbred homozygotes) of the balancers. Lindsley and Zimm 1992). The balancer chromosomes For each of the ten different inbred populations two sets of contain a series of overlapping inversions that prevent three replicate bottles were established and one set was successful recombination along the chromosome and con- placed at 25°C and the other at 29°C. In the first generation sequently the chromosome is inherited to next generation we observed problems with maintaining the populations at as a unit. In addition, both balancers carry a recessive 29°C probably due to a high incidence of male sterility that embryonic lethal, meaning that homozygotes for each of can occur at this temperature (Rohmer et al. 2004). the balancers do not develop. Therefore, the offspring of the bottles at 25°C were By the crossing procedure outlined in Fig. 1 we pro- transferred twice to new bottles for egg laying and one duced females that were heterozygous for both balancers in replicate was placed at a somewhat lower stress tempera- an otherwise inbred genetic background (except for the ture of 28.5°C. 123 452 Conserv Genet (2010) 11:449–462

Prior to the experiment, the ten inbred populations, and The rescue experiment is outlined in Fig. 2. It was ini- two additional inbred populations that were in the end not tiated by combining ten virgin females of either R12 or used for the experiments described here, were intercrossed R15 with 40 virgin females and 50 males of each of the for at least ten generations to produce a large outbred MIX remaining 8 inbred populations to start bottle populations population that served as a control population. For this with an initial frequency of 10% of the rescue genome, the population, ten replicate bottle populations were initiated so called rescued populations. For each combination of with a frequency of 5% of both balancers at the start of the rescuer and inbred population three replicates were estab- experiment and maintained in a similar way as the inbred lished at 25°C. From these bottles, the offspring were populations. For all populations and at both temperatures, the transferred to new bottles as parents for the next generation balancer frequencies were determined for eight generations. every fortnight. The original populations, further denoted The rationale behind this experiment is outlined by Sved as non-rescued populations, were maintained as single and Ayala (1970) and has been successfully applied by populations at the same time (for each population five Bijlsma et al. (1999). In short, balancer homozygotes are bottles that were mixed every generation). lethal and the wildtype homozygotes may also have a To assess the effect of immigration, viability (egg-to- decreased fitness because of inbreeding depression. The adult survival) was determined for the rescued and non- latter, therefore could be at a disadvantage compared to the rescued populations five and ten generations after the res- balancer heterozygotes. If the overall fitness of the balancer cue event (Fig. 2). From each replicate population five is greater than that of both homozygotes (the classical vials with 50 eggs each were established and viability was overdominance model) then the balancer heterozygote can calculated as the fraction of the eggs that resulted in adult introgress in the rescued population and will reach a stable flies. For the non-rescued populations ten vials with 50 equilibrium in the population. The frequency ultimately eggs each were initiated to determine viability. For each reached, depends on the degree of inbreeding depression replicate rescued population, the rescue effect was calcu- displayed by the inbred homozygotes. lated as being the average viability of the rescued replicate population minus the average viability of its non-rescued

counter part (Rescue effect = VRes - VNon-R). Note, that Rescue experiment we had a single viability estimate for each non-rescued population that was used for all three replicates of the In a separate experiment we studied the effect of immi- corresponding rescued populations. The rescue effect was gration on the viability of the original inbred populations determined for the populations both at 25 and at 29°C. using females of the inbred populations I12 and I15 as To determine to what extent the rescue event had affected immigrants to ‘‘rescue’’ the remaining eight inbred popu- the resistance to the high temperature stress, we additionally lations (as such I12 and I15 from then on are denoted as calculated the cost of stress. This is defined as the average R12 and R15, respectively). These two populations were viability of a population at 25°C compared to its average chosen as rescuers as they were found to be fixed for a viability at 29°C (Cost of stress = V25°C - V29°C). recessive conditional lethal system that causes almost 100% mortality in the pupal stage at 29°C but has no or little effect on pupal survival at 25°C (see ‘‘Results’’ sec- Pupal mortality after renewed inbreeding tion for details). Such conditional lethals seem to be present in many Drosophila species and populations (Do- After generation ten, the consequence of renewed bzhansky et al. 1955; Tobari 1966; Vermeulen and Bijlsma inbreeding on pupal mortality was assayed for all rescued 2004a, b; Bakker et al. 2010) and have been observed and non-rescued populations. To this end for each popu- before in low frequencies in the Groningen’83 base pop- lation (both rescued and non-rescued) twenty independent ulation (unpublished data). From this we infer that these pairs of flies were initiated and their offspring were full-sib conditional lethals are mildly deleterious at 25°C and mated for six additional generations, this time without maintained in a mutation-selection balance in this base replication (Fig. 2). One has to realize that for the non- population. As the rescue experiment and the concurrent rescued populations these six generations of inbreeding renewed inbreeding experiment are done at 25°C, we can came on top of the four generations they already had follow the increase of the frequency of the conditional le- undergone previously. During this process many of the thals by testing pre-adult or pupal survival at both 25 and full-sib lines became extinct before they reached the 29°C: an increase in the frequency of the lethal will result desired level of inbreeding (for population I5 none made it in an increase in pre-adult mortality at 29°C, even though a till the last generation). Consequently, the number of rep- small part of this increase might be attributed to the licate full-sib lines varied greatly among the populations. increase in inbreeding level (see ‘‘Results’’). After the inbreeding process was finished, of each inbred 123 Conserv Genet (2010) 11:449–462 453

Population i Population i Population i Rescue Non-rescued Rescued by R12 Rescued by R15 event

Viability test Viability test Viability test Test gen. 25oC and 29oC 25oC and 29oC 25oC and 29oC 5

Viability test Viability test Viability test Test gen. 25oC and 29oC 25oC and 29oC 25oC and 29oC 10

6 generations inbreeding Population i …. …. …. Non-rescued Full-sib (N-R; N-I) (N-R; I) (R12; I) (R15; I) lines

Pupal mortality test Pupal mortality test Pupal mortality test Pupal mortality test Test 25oC and 29oC 25oC and 29oC 25oC and 29oC 25oC and 29oC inbred lines

Fig. 2 Flow diagram of the rescue experiment and the concurrent the rescue event. Six generations inbreeding: from the rescued and renewed inbreeding. The different time points are: Rescue event gives non-rescued populations inbred lines were established after the tenth the parental generation in which immigrant females were combined generation by 6 generations of full-sib mating. Test inbred lines: pupal with each of eight inbred populations. Test generation 5: the rescue mortality was determined of all extant inbred lines after the sixth effect was determined ﬁve generations after the rescue event. Test generation of inbreeding. This was also done for the original inbred generation 10: the rescue effect was determined ten generations after populations

line two vials were initiated with 100 eggs each, one vial Models were fitted using a maximum likelihood approach was placed at 25°C and the other at 29°C. For each vial the and significance of fixed effects was assessed by means of total number of pupae and the number of pupae that did not log-likelihood ratio (v2) tests (Crawley 2007). We started eclose was determined and pupal mortality was calculated model building with full models including interactions of as the fraction of pupae that did not eclose. The cost of all orders, using backward elimination to arrive at final pupal stress is defined as the pupal mortality at 29°C minus models. The normality assumption was checked by visual the mortality at 25°C. For each rescued and non-rescued inspection of residuals of final models and by using QQ population the average over all full-sib lines was calcu- plots. No strong deviations from normality were observed. lated. At the same time the ten original inbred populations For the pupal mortality assay, we did not develop the full that had not undergone any further manipulation were also model as the design was unbalanced. Therefore, the effects assayed for pupal mortality at both temperatures (ten rep- of recurrent inbreeding, rescued versus non-rescued popu- licates with 100 eggs per vial for each population) and the lations, and R12 versus R15 were analysed separately. cost of pupal stress was calculated for these populations also (Fig. 2). Results Data analysis Balancer introgression experiment For the rescue and pupal mortality experiment, viabilities and mortality rates were modeled with linear mixed mod- Figure 3 shows the change in frequency of the balancer els, using the linear mixed effects (lme) procedure from the chromosomes at the three different treatments. At 25°C nlme package in R 2.9.0 (R Development Core Team (Fig. 3a–b) the balancers do increase in frequency in two of 2005). All effects were included as fixed effects, except the ten inbred populations. For I1 the frequency of the CyO ‘‘population’’ which was included as a random effect. balancer increases to a value higher than 0.25, which

123 454 Conserv Genet (2010) 11:449–462

Fig. 3 Changes in frequency 0.30 0.30 during eight generations of the A B 2nd chromosome balancer CyO 0.25 0.25 (left) and 3rd chromosome 0.20 0.20 -balancer

balancer TM3 (right) at three 3 temperatures 25°C(a–b), 0.15 0.15 28.5°C(c–d) and 29°C(e–f) when introduced in ten different 0.10 0.10 inbred populations. Specifically 0.05 0.05 marked populations are: I1 frequency Cy-balancer frequency TM (crosses), I5 (diamonds), I12 0.00 0.00 (open circles) and I15 (open 02468 02468 squares). The other six 0.6 0.6 populations are marked by dots. C D The outbred MIX population is 0.5 0.5 marked by triangles. Note, the scaling of the y axis in the top 0.4 0.4 -balancer figures is different from that of 3 the other figures 0.3 0.3 0.2 0.2

0.1 0.1 frequency Cy-balancer frequency TM 0.0 0.0 02468 02468

0.6 0.6 E F 0.5 0.5

0.4 0.4 -balancer 3 0.3 0.3

0.2 0.2

0.1 0.1 frequency TM

frequency Cy-balancer 0.0 0.0 02468 02468 generations generations means that 50% of the flies are in fact heterozygous for this population indicating the wildtype flies have again sub- balancer. No increase for the TM3 balancer is observed in stantial higher fitness than the balancer heterozygotes. For I1. Thus most inbreeding in this population can be attrib- the inbred populations we see now much higher frequen- uted to deleterious genes at the 2nd chromosome. For I5 cies for the balancers for most populations, particularly for both balancers reach frequencies higher than the starting the 3rd chromosome. Qualitatively the picture is similar for frequency, although the increase is less than observed for both temperatures, although there are somewhat larger I1, indicating that in this inbred population both the 2nd fluctuations due to small population size at 29°C where and the 3rd chromosome contribute to the inbreeding almost 40% of the (replicate) populations went extinct depression. before reaching the 8th generation. If we focus on the TM3 In contrast, the balancers are very rapidly lost in the balancer, we see that under temperature stress this balancer outbred MIX population and in generation 3 the CyO reaches appreciable frequencies in seven out of ten popu- balancer has disappeared from this population and TM3 is lations. To quantify the differences in frequency between found only in one replicate population at a frequency of outbred MIX and the inbred populations, we calculated the 0.0043. For most other inbred populations the frequency of average frequency of the balancer in generation five. For both balancers is also decreasing but at a clearly slower MIX both balancers had disappeared from all ten replicate rate. Nevertheless, in two out of ten populations the force populations at both 28.5 and 29°C. For the inbred popu- of heterosis is sufficiently large to facilitate introgression of lations, the averages (±SE) over all extant populations at even a recessive lethal. these temperatures for CyO were 0.047 ± 0.020 and When the temperature is increased to 28.5°C (Fig. 3c–d) 0.100 ± 0.035 at 28.5 and 29°C, respectively. For TM3 or 29°C (Fig. 3e–f) the picture changes considerably. Still these values were 0.162 ± 0.060 and 0.236 ± 0.061, the balancers disappear rapidly from the outbred MIX respectively. When calculated for the other generations

123 Conserv Genet (2010) 11:449–462 455

(from generation 3 onwards) the results are qualitatively Table 1 Results of the statistical analysis using mixed linear models not different from the fifth generation (data not shown). as described in ‘‘Materials and methods’’ showing only the results for For two inbred populations I12 and I15, the TM3 bal- those variables that significantly affected the rescue effect ancer reaches extreme frequencies of nearly 50%. As the Variable Contrast v2-value df P balancer can only exist in heterozygotes this means that Non-rescued viability 0.611 11.47 1 0.00071 (nearly) all flies present in the populations are heterozy- Temperature stress (29°C) -0.058 32.75 1 \10-7 gous and consequently, that at these temperatures homo- Rescuer R15 -0.049 21.80 1 \10-6 zygous inbred flies for the two populations do not exist. Temp. 9 rescuer -0.060 4.23 1 0.0397 Thus, populations I12 and I15 seem to be fixed for one or a few loci at the 3rd chromosome that cause lethality at high temperature. As this effect is not observed at 25°C (see ) Fig. 3, top), we can only conclude that these two popula- 0.4 tions are fixed for a conditional lethal (or lethal complex) Non-R 0.3 that is only expressed under high temperature stress. - V 0.2 Res Rescue experiment 0.1 0.0

From the previous experiment it is clear that I12 and I15 -0.1 show interesting characteristics. Therefore, we used these populations as rescuers for the other eight inbred popula- -0.2 rescue effect (V tions to study the dynamics of genetic rescue and to I1 I3 I5 I6 I7 I8 I11 I17

determine the rescue effect at both 25 and 29°C. The ) results are summarized in Fig. 4 and Table 1. 0.4 At 25°C there is a significant increase in viability by both Non-R 0.3 rescuers (Fig. 4, black bars) in both generation 5 and 10. It - V increases the viability on average by 7.5–15%. We observe 0.2 little difference between the two time points (Fig. 4) and Res 0.1 our statistical analysis showed there was no effect of gen- 0.0 eration on the rescue effect. This indicates that the rescue effect is not transient but persists for at least 10 generations. -0.1 However, there is significant variation between the different -0.2 rescue effect (V inbred populations with respect to the rescue effect. Fig- I1 I3 I5 I6 I7 I8 I11 I17 ure 5 clearly shows this variation for individual rescued populations in generation 5 (the picture for generation 10 is Fig. 5 Variation in the rescue effect among inbred populations five similar, not shown) for both rescuers. First of all, the vari- generations after the rescue event for the two rescuers R12 (top) and R15 (bottom). The values of the individual replicates are denoted by ation among replicates within populations is generally small filled circles, while the bars show the average over these replicates

) 0.20 compared to the variation among populations. There are a few exceptions for R15 (rescued populations I5, I6 and I7) 0.15

Non-R for which one replicate differs considerably from the other two. The reason for this is unclear at the moment. At the - V 0.10 population level we observe large differences between

Res 0.05 individual inbred populations. Focusing on R12 we see that 0.00 I1 shows an increase in viability around 30–40%, whereas I11 shows a non-significant increase of only a few percent. -0.05 For R15 the picture is similar although the rescue effect is -0.10 not as large as for R12. rescue effect (V R12-F5 R15-F5 R12-F10 R15-F10 Table 1 shows that the non-rescued viability has a highly significant effect on the increase in viability after Fig. 4 The average rescue effect (±SE) for the two rescuer lines R12 the rescued event. This is illustrated in Fig. 6 that shows and R15 five generations (F5) and ten generations (F10) after the rescue event. The black bars show the rescue effect at 25°C and the the relation between non-rescued viability and the rescue grey bars the effect at 29°C effect in both generation five and ten. We observe a strong 123 456 Conserv Genet (2010) 11:449–462

) those rescued by R15, while R12 did not differ signiﬁ- R-line 12 0.4 cantly from both of these in both generations. R-line 15 Non-R Figure 4 also indicates a difference between the rescu- - V 0.2 ers: R15 shows on average a smaller rescue effect than

Res R12. In agreement, our statistical analysis shows a significant effect of the rescuer with respect to the rescue effect 0.0 as well as a signiﬁcant interaction between rescuer and temperature (Table 1). Basically, R15 seems to be less -0.2 effective at 25°C (Fig. 4, black bars) but at the same time

rescue effect (V populations rescued by R15 are more strongly affected by 0.4 0.5 0.6 0.7 0.8 0.9 1.0 the stress temperature (Fig. 4, grey bars). We expected that the rate of introgression of the lethal might be positively

-R) R-line 12 0.4 R-line 15 correlated to the size of the rescue effect. However, the

Non correlation between these two factors turned out to be not -V 0.2 signiﬁcant at both generation 5 and 10 (data not shown). Res Recurrent inbreeding and pupal mortality 0.0 The increased cost of stress after the rescue event is most -0.2 probably due to introgression of the conditional lethal into

rescue effect (V the rescued populations at the benign temperature. To 0.30.40.50.60.70.80.91.0 investigate the fate and consequence of such (a) gene(s) non-rescued viability under renewed inbreeding, we tested the difference in pre- Fig. 6 Relation between the rescue effect and non-rescued viability adult mortality of inbred lines from both the rescued and for the two rescuer lines in generation F5 (top) and F10 (bottom) and non-rescued populations. As it was at that time clear that the the linear regression through these for R12 (black) and R15 (grey) conditional lethal caused specifically mortality in the pupal stage at 29°C only (and not at 25°C), we used pupal mor- negative relationship showing that rescuing is more suc- tality as measure instead of pre-adult mortality. As a broad cessful when the non-rescued viability is lower. picture, Fig. 7a shows the increase in pupal mortality at As R12 and R15 both carry a conditionally expressed 29°C compared to 25°C averaged over 7 inbred populations recessive lethal that is mildly deleterious at 25°C and (no inbred lines from population I5 did survive the 6 gen- lethal at 29°C, introgression of this lethal at 25°C (the erations of recurrent full-sib mating). This is shown for the temperature the rescue experiment was done) will affect different types of populations in the experiment (see Fig. 2): the rescue effect at 29°C. Figure 4 shows that indeed the the original populations non-rescued populations (N-R;N- rescue effect decreases when the rescued populations are I), the non-rescued populations after six additional rounds tested at this high temperature (grey bars). Table 1 shows of full-sib mating (N-R;I), and the rescued populations after that this effect is highly significant. To show that the additional inbreeding (R12;I and R15;I). As the experi- rescued populations have become more sensitive to high mental design was quite unbalanced (different degrees of temperature stress we calculated the cost of stress being replication at different levels), we analysed the data for the the difference in viability between 25 and 29°C averaged different factors separately with mixed linear models. First over all populations. In generation 5 means (±SE) were we tested for the effect of the additional inbreeding on pupal 0.151 ± 0.023, 0.203 ± 0.025 and 0.280 ± 0.048 for non- mortality and found a significant effect (v2 = 4.49, df = 1, rescued, rescued by R12 and rescued by R15, respectively. P = 0.034). Comparing N-R;N-I with N-R;I shows that the In generation 10 the data were 0.114 ± 0.033, 0.160 ± 6 generations of additional inbreeding more than doubles 0.024 and 0.234 ± 0.027 for non-rescued, rescued by R12 the level of pupal mortality (Fig. 7a). This indicates that and rescued by R15, respectively. In both generations inbreeding per se increases pupal mortality at temperature the cost of stress is indeed the lowest for the non-res- stress. Second, the comparison between additionally inbred cued populations and the highest for populations rescued non-rescued populations (N-R;I) and additionally inbred by R15. A one-way analysis of variance revealed that the rescued populations (R12;I and R15;I) showed that rescuing differences are significant both in generation 5 (F2,23 = did significantly increase the pupal mortality at 29°C 2 3.68, P = 0.04) and generation 10 (F2,23 = 4.74, (v = 22.52, df = 1, P \ 0.0001). As the inbreeding level P = 0.02). A multiple comparison of means showed that is the same for all populations this indicates that the the non-rescued populations differed significantly from increase is due to the introgression of the conditional lethal 123 Conserv Genet (2010) 11:449–462 457

A rescue effect of R15 decreases much more under tempera- 0.4 ture stress compared to R12 (Fig. 4). At the level of the individual populations, we observed 0.3 signiﬁcant variation both among and within populations (Fig. 7b–c). Figure 7b shows the variation in the increase 0.2 in pupal mortality among lines for the non-rescued compared to the rescued populations. This ﬁgure again illus- 0.1 trates clearly the difference between the rescued and non- rescued populations and also that the mortality in general is

increase in pupal mortality 0.0 higher for R15 than for R12. The exception is population I8 N-R; N-I N-R; I R12; I R15; I that shows a relatively high level of pupal mortality as non- rescued population, but does not show any increase in 0.5 B mortality after the rescue event. The within population variation is shown for a few 0.4 selected cases in Fig. 7c. It is clear that for I17 and I1 the 0.3 individual inbred lines made from these populations ten generations after the rescue event show a highly variable 0.2 effect on pupal mortality. For some inbred lines pupal mortality does not increase at 29°C compared to 25°C, 0.1 while for others it increases to 100% pupal mortality. In

increase in pupal mortality 0.0 fact, for these two populations we see a bi-model or near I1 I3 I6 I7 I8 I11 I17 uniform distribution of the increase in pupal mortality due to high temperature. This is compatible with the idea that 1.0 C the conditional lethal has introgressed to a reasonable high 0.8 frequency in these populations and, as we did the inbreeding at 25°C, has become subject to severe genetic 0.6 drift during the six generations of full-sib matings leading to extreme frequencies in individual inbred lines. A con- 0.4 trasting picture is observed for population I6 for which the variation among inbred lines is relatively small, suggesting 0.2 that in this case the conditional lethal did not introgress into the population, or reached only very low frequencies, increase in pupal mortality 0.0 so that the applied genetic drift did not cause variation 17-R12 17-R15 6-R12 6-R15 1-R12 1-R15 among lines. This seems to agree with our observation Fig. 7 Average increase in pupal mortality at 29°C compared to at concerning the size of the rescue effect for this population 25°C at three levels. a Shows the mean (±SE) averaged over all (Fig. 5). inbred populations for four different groups: N-R; N-I: original inbred populations, N-R; I: non-rescued populations but additionally inbred, R12; I: rescued by R12 and additionally inbred and R15; I: rescued by R15 and additionally inbred. b Shows the average (±SE) for the Discussion different populations tested for N-R; I (black bars), R12; I (light grey bars) and R15; I (dark grey bars). c shows the variation in the Introgression of deleterious alleles increase of pupal mortality among individual inbred lines for three inbred populations (I17, I6 and I1) rescued by either R12 or R15. The grey bars denote the average over the replicate inbred lines (filled Under benign conditions we observed that in eight out of black circles) ten populations the balancers disappeared from the rescued populations although at a slower rate than in the outbred MIX populations (Fig. 3). This means that inbred wildtype into the rescued populations. Third, we tested the differ- homozygotes have a lower fitness than wildtype flies of the ences due to the rescuers (R12 vs. R15) and found this outbred population, but the fitness loss is not such that that difference to be highly significant (v2 = 21.58, df = 1, it leads to a stable polymorphism in the populations and in P \ 0.0001). This could mean that the introgression of the the end the balancers are lost. This does not necessarily conditional lethal was more successful for R15 than for imply that there is no inbreeding depression in these pop- R12. This is not in line with our previous finding that R12 ulations because the inbred homozygotes have a similar or is a better rescuer than R15, but would explain why the larger fitness than the balancer heterozygotes. It is well 123 458 Conserv Genet (2010) 11:449–462 known that balancer heterozygotes have a significantly explains why the rescue effect is generally expected to be lower fitness than wildtype heterozygotes (Tracey and the strongest in the first generation(s) after a rescue event Ayala 1974; Mackay 1985). This reduction in fitness has but is diluted in subsequent generations (Lynch 1991). been estimated to be around 20%. This means that even if Because of this, there is little chance that recessive, highly the inbreeding depression is not much less than 20% per deleterious alleles from the immigrant genome would reach chromosome, the balancer will still disappear from the appreciable frequencies in rescued population like inbred population. observed in our balancer experiment, unless it is tightly At the same time we observed that under thermal stress linked to a locus with a very strong heterotic effect. the balancers reached much higher frequencies and per- However, mildly deleterious alleles, probably responsible sisted at appreciable frequencies in most of the rescued for the greater part of the inbreeding depression observed populations, particularly so for the TM3 balancer (Fig. 3). in inbred populations (Hedrick 1994), still are expected to Consequently, these populations suffer considerably more be able to increase substantially in frequency when linked from inbreeding depression under stress than under benign to a locus causing the heterotic effect. conditions. This can only be explained assuming that, That hitchhiking can be responsible for an increase of compared to 25°C, under stress the fitness of the inbred alleles from the immigrant into the rescued population is wildtypes decreases relative to that of the balancer het- clearly shown in our pupal mortality experiment. As erozygotes. In other words, inbreeding depression increases mortality during the pupal stage is the specific phenotype under the applied temperature stress in most inbred popu- of lethal homozygotes, we can assume that the increase in lations. This agrees well with previous findings for this pupal mortality at 29°C in the rescued populations com- population (Bijlsma et al. 1999) and adds to an increasing pared to the non-rescued populations (Fig. 7b) is mainly number of data showing that the negative effects of due to the increased frequency of the conditional lethal that inbreeding greatly increase under stress conditions was present in the rescuers R12 and R15. From this, we (Dahlgaard and Hoffmann 2000; Keller et al. 2002; Am- infer that this lethal must have reached substantial fre- bruster and Reed 2005). quencies in most rescued populations. Also the data on the The balancer experiment showed that the heterosis increase of the cost of stress support this finding. However, accompanying genetic rescue can be a powerful force to under the temperature conditions the rescued populations allow even highly deleterious alleles, in this case a reces- were maintained (25°C), the lethal effect is not expressed sive lethal, to increase in frequency in the rescued popu- and the lethal allele is most probably mildly deleterious. lations. This force is particularly strong when the fitness of So, an increase in frequency of the conditional lethals can the rescued population is low, as I1 and I5 were among the only be explained by hitchhiking along with the loci that populations showing the highest inbreeding depression for are under selection and responsible for the heterotic effect. viability. Most extreme was the situation for inbred pop- This is in agreement with the finding that rescue events ulations I12 and I15 at 29°C where only balancer hetero- often show a strong increase in the heterozygosity for zygotes were viable under these conditions and the neutral microsatellites (Madsen et al. 1999; Bouzat et al. recessive lethal carried by the immigrants did reach a 2009). frequency of 0.50 in the rescued populations. Thus when In conclusion, there is little doubt that genetic rescue of deleterious alleles from the immigrants are tightly linked to greatly inbred populations might cause a disproportional the genes that are responsible for the heterosis and mask increase in the frequency of mildly deleterious alleles that inbreeding depression, this deleterious gene can hitchhike are carried by the immigrants. along and reach appreciable frequencies in the recipient Can we prevent this to happen by carefully selecting the population. most fit individuals of a donor population as immigrants? For the increase in frequency of the deleterious alleles The answer will be no. Assuming that inbreeding depression linkage to the genes that are responsible for the heterosis is for the greater part is due to homozygosity for recessive essential. In that light, our balancer experiment is some- deleterious alleles, species that suffer from high inbreeding what artificial as both the entire 2nd and 3rd chromosome depression when confined to small population size neces- each form one linkage group. Consequently, the heterotic sarily are species that carry a high amount of lethal equiv- force of all loci at a balancer chromosome that contribute alents. Half of these lethal equivalents are thought to be to heterosis are combined in their effort and inseparable. caused by many loci that have small fitness effects For inversion free chromosomes, recombination would (Charlesworth and Charlesworth 1999; Lynch et al. 1999). uncouple these loci in later generations whereby the het- As a consequence, all individuals of those species that erotic force becomes smaller as individuals will be het- display high levels of inbreeding depression upon inbreed- erozygous for only a subset of the loci that are responsible ing are expected to carry part of this load in heterozygous for the heterosis in the first generation hybrids. This condition and, as such, escape the scrutiny of selection. 123 Conserv Genet (2010) 11:449–462 459

The rescue effect an immigrant male and showed that heterosis was the driving force. Our method, introducing virgin females into Our data show that genetic rescue can be effective to the resident populations resembles the latter experiment increase the fitness of inbred populations, particularly when and we conclude that heterosis is also the driving force in the non-rescued fitness was low (Fig. 6). This is, of course, our case. Based on the findings that the rescue effect not unexpected as it is impossible to improve the viability decreased significantly when tested at 29°C (Fig. 4) and the of populations that have already a viability near the max- observed high pupal mortality at the same temperature imum. The viability assay used here, normally reveals a (Fig. 7), we infer that the conditional lethal, and by viability between 0.75 and 0.90 for fully outbred popula- extrapolation also other parts of the immigrant genome, tions. This implies that the inbred populations that showed had attained appreciable frequencies in most of the rescued a viability above 0.75 when non-rescued will show very populations. This agrees well with the results of Saccheri little gain from genetic rescue. In fact, if we add the rescue and Brakefield (2002). effect to the non-rescued viability (see Fig. 6) we find that The rescue effect also depended significantly on the most rescued populations have attained a viability of 0.75 rescuer used. R12 increased the fitness of the rescued or more after the rescue event. This means that they all populations to a higher level than R15. On the other hand, reached a viability (nearly) similar to that of outbred the conditional lethal seems to have introgressed to a sig- populations. This suggests that the observed negative cor- nificant higher frequency in the rescued population when relation between the non-rescued viability and the rescue R15 was used than using R12. This can be inferred from effect is for the greater part due to the methodology. the fact that the inbred populations rescued by R15 showed As we were interested in the more long-term conse- a larger decrease in the rescue effect (Fig. 4) and a higher quences of genetic rescue, we determined the rescue effect increase in pupal mortality (Fig. 7) when tested at 29°C. It 5 and 10 generations after the rescue event and observed is conceivable that the two rescuer populations differed that the increase in fitness persisted during this interval. As considerably in genetic make up (it is at the moment even such, we do not know the dynamics during the first gen- unknown if the conditional lethals are the same). Another eration directly after the rescue event. It could be that the important factor in this respect is that we focused on a fitness increase due to immigration was larger during the single fitness component, pre-adult viability. However, first generations and then declined to the value we observed many other fitness components in Drosophila show high at generation 5. This would agree with the idea that the levels of inbreeding depression upon inbreeding (Lynch heterosis effect becomes diluted in later generations and Walsh 1998; Kristensen and Sorensen 2005; Kristen- (Lynch 1991; Hooftman et al. 2007). On the other hand, if sen et al. 2010). It might well be that some of these, like the population size of the recipient population is not too fecundity or sterility, also showed strong heterosis upon small, the number of heterozygotes may be expected to genetic rescue and affected the introgression of the immi- increase over the first consecutive generations and the fit- grant genome. To evaluate the success of genetic rescue ness could have steadily climbed to the value observed in more fully it is necessary to study many more fitness generation 5. Whatever, our data show that the rescue components. effect is not a short-lived and transient phenomenon but Notwithstanding, these uncertainties, our results show persists for longer periods, at least when population sizes that genetic rescue is successful, like found in other cases, are kept reasonably high after the rescue event. and that the positive effect on fitness is not transient but A difference between our approach and what will be persists over longer periods. This agrees well with the normal practice in managing endangered populations is findings of Willi et al. (2007). that we used individuals from another highly inbred population as immigrants. To what extent this did affect the Recurrent inbreeding outcome of the experiment is not clear at the moment. However, whereas Ball et al. (2000) showed that outbred Genetic rescue is applied as a management measure to individuals performed significantly better than inbred overcome the decrease in fitness of small isolated popula- immigrants, Saccheri and Brakefield (2002) showed that tions due to genetic drift and inbreeding. However, the even highly inbred immigrants performed quite well as long-term benefits of this approach are only warranted if it rescuer. The outcome may also depend on how the rescue is accompanied by a substantial increase in population size. was done. Ball et al. (2000) used a single male of immi- If the population stays relatively small and genetic drift and grant that had to compete with other males for access to the inbreeding are still significant forces, the fitness is expected females and found that the vigour of the immigrant male to decrease again in subsequent generations (Liberg et al. was decisive for the outcome. Saccheri and Brakefield 2005; Robert et al. 2007). Even worse, as mildly deleteri- (2002) started with one resident female that was mated to ous alleles present in the immigrants might have reached 123 460 Conserv Genet (2010) 11:449–462 appreciable frequencies in the rescued populations, immigrants contribute equally to the next generations, the renewed inbreeding and genetic drift may lead to even increase of an allele present in one out of ten immigrants is more severe inbreeding depression. This is due to the fact expected to reach an average frequency of only 10% of that that, assuming the deleterious alleles present behave near when this individual was used as a single immigrant. neutral under genetic drift (Ns \ 1), the fixation probability Although introducing ‘‘many’’ immigrants may threaten is directly related to the frequency the deleterious alleles the genetic integrity of the recipient population somewhat, had attained in the rescued population: the probability of it will also prevent a reduction of Ne (see Hedrick and fixation is the same as the initial allele frequency (Kimura Fredrickson 2010) as none of the immigrant genomes will 1983). Even in a situation where natural selection is partly become predominant in the rescued population. If for other effective, still a higher frequency of the deleterious alleles reasons the use of few immigrants is preferable, it is rec- would mean a higher probability of fixation. ommended to repeat the immigration of unrelated immi- We have mimicked this process by subjecting the res- grants in the subsequent generations to prevent certain cued populations to renewed inbreeding. The conditional immigrant genomes, and thereby also its recessive mildly lethal(s) from both rescuers will have reached substantial deleterious alleles, to become overrepresented. frequencies while hitchhiking along with the genes A second issue is that genetic rescue necessarily has to responsible for the heterosis (see Fig. 7). Because the be accompanied by demographic and environmental mea- conditional lethals are mildly deleterious or nearly neutral sures (see Robert et al. 2007). If population size is only at 25°C, the temperature at which the inbreeding was done, temporarily boosted and genetic drift and inbreeding take the deleterious alleles will be subject to genetic drift and precedence again in later generations, the situation rapidly can reach high frequencies in individual inbred lines or can become worse than before the rescue event. This even go to fixation (Fig. 7c). When such lines are subse- means that transplantation of individuals to endangered quently tested at 29°C they show low survival and readily populations only makes sense when habitat alterations and will go extinct. increasing connectivity warrant that the population size is The same process is expected to affect other mildly permanently boosted. If not, genetic rescue has to be reg- deleterious alleles that were present in the rescuers. Thus, ularly repeated. initially successful genetic rescue followed by subsequent Although there are quite a number of risks connected inbreeding and genetic drift in many cases is expected to with genetic rescue (Tallmon et al. 2004; Edmands 2007; lead to even more severe loss of fitness in the rescued Hedrick and Fredrickson 2010) including the ones dis- population than it originally experienced before the rescue cussed here, genetic rescue has proven to be a successful event. method to increase the fitness of genetically eroded populations and to improve their persistence (Hedrick 1995; Inferences for conservation management Westemeier et al. 1998; Madsen et al. 1999; Willi et al. 2007; Bouzat et al. 2009; Hedrick and Fredrickson 2010). In this paper we evaluated the dynamics of genetic rescue Thus if populations decline in numbers partly due to and show that the positive aspect of genetic rescue is a genetic problems and because of this have a high proba- long-term increase in fitness of the rescued population. bility of going extinct in the near future, genetic rescue However, this will be accompanied by an increase in the offers a viable management option, notwithstanding the frequency of recessive deleterious alleles carried by the risks connected to it. immigrants, which, as the population experiences recurrent inbreeding and drift again, may endanger persistence of the Acknowledgments We thank Phil Hedrick, Torsten Kristensen and rescued population even more. However, this will only be a an anonymous reviewer for valuable suggestions to improve earlier versions of the manuscript. We thank Anneke Boerema, Herve´ Rohier problem when the deleterious alleles present in the rescuers and Rogier Houwerzijl for technical assistance. Part of the work was reach reasonable high frequencies. This can be overcome done while RB was visiting the stress group of Volker Loeschcke that by using a sufficient number of unrelated immigrants and was made possible by a grant from the University of Aarhus (DK). ensure that they all contribute more or less equally to the This work has greatly profited from discussions within the framework the Research Networking Programme ConGen funded by the Euro- next generations. As there are many loci in large popula- pean Science Foundation. tions that carry deleterious alleles but these generally have low frequencies, the immigrants will all carry deleterious Open Access This article is distributed under the terms of the alleles but mostly different ones. Although these still may Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any increase in frequency due to the force of heterosis, the medium, provided the original author(s) and source are credited. increase will be much lower for individual alleles. If all

123 Conserv Genet (2010) 11:449–462 461

References relative hybridization: a case study using four generations of hybrids. J Appl Ecol 44:1035–1045 Ambruster P, Reed DH (2005) Inbreeding depression in benign and Keller LF, Waller DM (2002) Inbreeding effects in wild populations. stressful environments. Heredity 95:235–242 Trends Ecol Evol 17:230–241 Bakker J, Van Rijswijk MEC, Weissing FJ, Bijlsma R (2010) Keller LF, Grant PR, Grant BR, Petren K (2002) Environmental Consequences of fragmentation for the ability to adapt to novel conditions affect the magnitude of inbreeding depression in environments in experimental Drosophila metapopulations. survival of Darwin’s finches. Evolution 56:1229–1239 Conserv Genet (this issue). doi:10.1007/s10592-010-0052-5 Kimura M (1983) The neutral theory of molecular evolution. Ball SJ, Adams M, Possingham HP, Keller MA (2000) The genetic Cambridge University Pres, Cambridge contribution of a single male immigrant to small, inbred Kristensen TN, Sorensen AC (2005) Inbreeding—lessons from populations: a laboratory study using Drosophila melanogaster. animal breeding, evolutionary biology and conservation genet- Heredity 84:668–677 ics. Anim Sci 80:121–133 Bijlsma R, Bundgaard J, Van Putten WF (1999) Environmental Kristensen TN, Pedersen KS, Vermeulen CJ, Loeschcke V (2010) dependence of inbreeding depression and purging in Drosophila Research on inbreeding in the ‘omics’ era. Trends Ecol Evol 25: melanogaster. J Evol Biol 12:1125–1137 44–52 Bijlsma R, Bundgaard J, Boerema A (2000) Does inbreeding affect Leducq JB, Gosset CC, Poiret M, Hendoux F, Vekemans X, Billiard S the extinction risk of small populations? Predictions from (2010) An experimental study of the S-Allele effect in the self Drosophila. J Evol Biol 13:502–514 incompatible Biscutelle neustriaca (Brasicaceae). Conserv Genet Bouzat JL, Johnson JA, Toepfer JE, Simpson SA, Esker TL, (this issue). doi:10.1007/s10592-010-0055-2 Westemeier RL (2009) Beyond the beneficial effects of trans- Liberg O, Andreń H, Pederson H-C, Sand H, Sejbeg D, Wabakken P, ˚ locations as an effective tool for the genetic restoration of Akesson M, Bensch S (2005) Severe inbreeding depression in a isolated populations. Conserv Genet 10:191–201 wild wolf (Canis lupus) population. Biol Lett 1:17–20 Caughley G (1994) Directions in conservation biology. J Anim Ecol Lindsley DL, Zimm GG (1992) The genome of Drosophila melano- 63:215–244 gaster. Academic Press, San Diego Ceballos G, Ehrlich PR (2002) Mammal population losses and the Lynch M (1991) The genetic interpretation of inbreeding depression extinction crisis. Science 296:904–907 and outbreeding depression. Evolution 45:622–629 Charlesworth D, Charlesworth B (1999) The genetic basis of Lynch M, Walsh B (1998) Genetics and analysis of quantitative traits. inbreeding depression. Genet Res 74:329–340 Sinauer Associates, Sunderland Crawley MJ (2007) The R Book. Wiley, Chichester Lynch M, Blanchard J, Houle D, Kibota T, Schultz S, Vassilieva L, Dahlgaard J, Hoffmann AA (2000) Stress resistance and environ- Willis J (1999) Perspective: spontaneous deleterious mutations. mental dependency of inbreeding depression in Drosophila Evolution 53:645–663 melanogaster. Conserv Biol 14:1187–1192 Mackay TFC (1985) A quantitative genetic analysis of fitness and its Dobzhansky T, Pavlovsky O (1958) Interracial hybridization and components in Drosophila melanogaster. Genet Res Camb 47: breakdown of coadapted gene complexes in Drosophila paulis- 59–70 torum and Drosophila willistoni. Proc Natl Acad Sci USA 44: Madsen T, Shine R, Olsson M, Wittzell H (1999) Restoration of an 622–629 inbred adder population. Nature 402:34–35 Dobzhansky T, Pavlovsky O, Spassky B, Spassky N (1955) Genetics Maynard Smith J, Haigh J (1974) The hitchhiking effect of a of natural populations. XXIII. Biological role of deleterious favourable gene. Genet Res 23:23–35 recessives in populations of Drosophila. Genetics 40:781–796 Newman D, Pilson D (1997) Increased probability of extinction due Ebert D, Haag C, Kirkpatrick M, Riek M, Hottinger JW, Pajunen VI to decreased genetic effective population size: experimental (2002) A selective advantage to immigrant genes in a Daphnia populations of Clarkia pulchella. Evolution 51:354–362 metapopulation. Science 295:485–488 R Development Core Team (2005) R: a language and environment for Edmands S (2007) Between a rock and a hard place: evaluating the statistical computing. R Foundation for Statistical Computing, relative risks of inbreeding and outbreeding for conservation and Vienna, Austria management. Mol Ecol 16:463–475 Robert A, Couvet D, Sarrazin F (2007) Integration of demography Falconer DS, Mackay TCF (1996) Introduction to quantitative and genetics in population restoration. Ecoscience 14: genetics, 4th edn. Pearson Education Ltd, Harlow, England 463–471 Frankham R (1995) Conservation genetics. Ann Rev Genet 29:305–327 Rohmer C, David JR, Moreteau B, Joly D (2004) Heat induced male Hedrick PW (1994) Purging inbreeding depression and the probability sterility in Drosophila melanogaster: adaptive genetic variations of extinction: fullsib mating. Heredity 73:363–372 among geographic populations and role of the Y chromosome. Hedrick PW (1995) Gene flow and genetic restoration: the Florida J Exp Biol 207:2735–2743 panther as a case study. Conserv Biol 9:996–1007 Saccheri IJ, Brakefield PM (2002) Rapid spread of immigrant Hedrick PW (2005) ‘‘Genetic restoration’’: a more comprehensive genomes into inbred populations. Proc R Soc B 269:1073– perspective than ‘‘genetic rescue’’. Trends Ecol Evol 20:109 1078 Hedrick PW, Fredrickson RJ (2010) Genetic rescue guidelines with Saccheri I, Kuussaari M, Kankare M, Vikman P, Fortelius W, Hanski examples from Mexican wolves and Florida panthers. Conserv I (1998) Inbreeeding and extinction in a butterfly metapopula- Genet (this issue). doi:10.1007/s10592-009-9999-5 tion. Nature 392:491–494 Hedrick PW, Kalinowski S (2000) Inbreeding depression and Spielman D, Brook BW, Frankham R (2004) Most species are not conservation biology. Ann Rev Ecol Syst 31:139–162 driven to extinction before genetic factors impact them. Proc Hedrick PW, Lacy RC, Allendorf FW, Soule´ ME (1996) Directions in Natl Acad Sci USA 101:15261–15264 conservation biology: comments on Caughley. Conserv Biol 10: Sved JA, Ayala FJ (1970) A population cage test for heterosis in 1312–1320 Drosophila melanogaster. Genetics 66:97–113 Hooftman DAP, Jong MJD, Oostermeijer JGB, Den Nijs HJCM Tallmon DA, Luikart G, Waples R (2004) The alluring simplicity and (2007) Modeling the long-term consequences of crop-wild complex reality of genetic rescue. Trends Ecol Evol 19:489–496

123 462 Conserv Genet (2010) 11:449–462

Tobari I (1966) Effects of temperature on the viabilities of a severely bottlenecked wolf (Canis lupus) population by a homozygotes and heterozygotes for second chromosomes in single immigrant. Proc R Soc B 270:91–97 Drosophila melanogaster. Genetics 54:783–791 Wang JL (2000) Effects of population structures and selection Tracey ML, Ayala FJ (1974) Genetic load in natural populations: is it strategies on the purging of inbreeding depression due to compatible with the hypothesis that many polymorphisms are deleterious mutations. Genet Res Camb 76:75–86 maintained by natural selection? Genetics 77:569–598 Wang J, Hill WG, Charlesworth D, Charlesworth B (1999) Dynamics Vermeulen CJ, Bijlsma R (2004a) Changes in mortality patterns and of inbreeding depression due to deleterious mutations in small temperature dependence of lifespan in Drosophila melanogaster populations: mutation parameters and inbreeding rate. Genet Res caused by inbreeding. Heredity 92:275–281 74:165–178 Vermeulen CJ, Bijlsma R (2004b) Characterization of conditionally Westemeier RL, Brawn LD, Simpson SA, Esker TL, Jansen RW, expressed mutants affecting age-speciﬁc Drosophila melanogas- Walk JW, Kershner EL, Bouzat JL, Paige KN (1998) Tracking ter: lethal conditions and temperature-sensitive periods. Genetics the long-term decline and recovery of an isolated population. 167:1241–1248 Science 282:1695–1698 Vermeulen CJ, Bijlsma R (2006) Changes in genetic architecture Willi Y, van Kleunen M, Dietrich S, Fischer M (2007) Genetic rescue during relaxation in Drosophila melanogaster selected on persists beyond the ﬁrst-generation outbreeding in small popu- divergent virgin life span. J Evol Biol 19:216–227 lations of a rare plant. Proc R Soc B 274:2357–2364 Vila` C, Sundqvist A-K, Flagstad Ø, Seddon J, Bjo¨rnerfeldt S, Kojola I, Casulli A, Sand H, Wabakken P, Ellegren H (2003) Rescue of

123