Is Mutation Accumulation a Threat to the Survival of Endangered Populations? DEAN M. GILLIGAN, LYNN M. WOODWORTH, MARGARET E. MONTGOMERY, DAVID A. BRISCOE, AND RICHARD FRANKHAM* Key Centre for Biodiversity and Bioresources, School of Biological Sciences, Macquarie University, NSW 2109, Australia
Abstract: The accumulation of new deleterious mutations has been predicted to constitute a significant threat to the survival of finite sexually reproducing populations. Three measures of genetic load were made on populations of Drosophila melanogaster maintained at effective population sizes of 25, 50, 100, 250, and 500 for 45 or 50 generations and their outbred base population and a new sample from the same wild population. Genetic loads were measured as fitness differentials between inbred and non-inbred lines derived from each population under both benign (productivity of single pairs) and competitive (competitive index) conditions. No trend of smaller populations exhibiting greater genetic loads than larger ones was observed under either benign or competitive conditions. Further, genetic loads were similar in captive and wild populations. Frequencies of deleterious and lethal alleles on chromosome II were measured by making the chromosome (approximately 40% of the genome) homozygous using a marked balancer stock. Neither deleterious nor lethal allele frequencies exhibited a relationship with population size. The accumulation of detrimental mutations does not appear to pose a significant threat to finite sexual populations with effective sizes of 25 or more over the 100–200 year time frames considered in most wildlife conservation programs. ¿Es la Acumulación de Mutaciones una Amenaza para la Supervivencia de Poblaciones en Peligro? Resumen: Se ha predicho que la acumulación de mutaciones deletéreas neuvas constituye una amenaza significativa para la supervivencia de poblaciones sexualmente reproductoras finitas. Se tomaron tres medidas de carga genética en poblaciones de Drosophila melanogaster mantenidas en tamaños poblacionales efectivos de 25, 50, 100, 250 y 500 por 45 o 50 generaciones y su población base no consanguínea y una muestra neuva de la misma población silvestre. Las cargas genéticas se midieron como diferencias de adaptabilidad entre líneas consanguíneas y no consanguíneas derivadas de cada población bajo condiciones benignas (productividad de parejas individuales) y competitivas (índice de competitividad). Bajo condiciones benignas y competitivas no se observó tendencia de las poblaciones pequeñas a presentar cargas genéticas mayores que las de poblaciones grandes. Más aun, las cargas genéticas fueron similares en las poblaciones cautivas y silvestres. Se midieron las frecuencias de aleolos deletéreos y letales en el cromosoma II haciendo homocigótico al cromosoma (aproximadamente 40% del genoma) mediante un patrón balanceador marcado. Las frecuencias de alelos delétereos y letales no mostraron relaciones con el tamaño poblacional. La acumulación de mutaciones perjudiciales parece no ser una amenaza significativa para poblaciones sexuales finitas con tamaños efectivos de 25 o más individuos en las escalas de 100–200 años consideradas en la mayoría de los programas de conservación de vida silvestre.
* Address correspondence to R. Frankham, email
[email protected] Paper submitted June 24, 1996; revised manuscript accepted December 6, 1996.
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Introduction The accumulation of new mildly deleterious mutations has been predicted to pose a serious extinction risk for finite sexual populations (Lande 1994; Lynch et al. 1995a, 1995b). Lynch and Gabriel (1990) termed this extinction process “mutational meltdown.” Lande (1994) predicted that mutation accumulation poses a serious threat to populations with effective sizes (Ne ) as large as 1000, whereas Lynch et al. (1995b) predicted that it would be a threat to populations smaller than Ne 5 100 and that it may cause extinction within 100 generations. Lynch et al. (1995b) suggested that these models underestimate the actual extinction risk posed by mutation accumulation. Deleterious mutations impose a load on populations by reducing survival and reproductive rates, thus increasing the likelihood of population extinction (Lynch & Gabriel 1990; Gabriel et al. 1991; Kondrashov & Turelli 1992; Charlesworth et al. 1993a; Gabriel et al. 1993; Lynch et al. 1993; Gabriel & Bürger 1994; Hedrick 1994; Lande 1995). In large populations natural selection is an efficient mechanism for removing new deleterious mutations, especially those with large effects (Lynch & Gabriel 1990; Gabriel et al. 1991; Lynch et al. 1993). As population size is reduced the efficiency of natural selection decreases (Charlesworth et al. 1993b), with alleles having a selection coefficient less than 1/2Ne being effectively neutral (Kimura 1983). Therefore, the fixation of increasingly deleterious alleles becomes more probable as the effective population size is reduced. Mutation accumulation and the subsequent loss of fitness has been observed in asexual populations (Chao 1990; Chao et al. 1992; Anderson & Hughes 1996; Kibota & Lynch 1996). Further, chromosomes of sexually reproducing species have been shown to accumulate deleterious mutations after many generations of maintenance without recombination (Mukai et al. 1972; Rice 1994; Vrijenhoek 1994). Consequently, the occurrence of mutation accumulation in obligately asexual populations is widely accepted. However, the persistence of ancient asexual lineages ( Judson & Normark 1996; Moran 1996) indicates that it may not affect all populations. In sexually reproducing organisms, recombination and segregation can result in the production of offspring that are fitter than their parents. This allows natural selection to remove mutations from their genomes (Lynch & Gabriel 1990). Prior to the work of Lande (1994) and Lynch et al. (1995a, 1995b), it was believed that sexual populations larger than a dozen individuals would rarely experience mutation accumulation to the point where their survival was endangered (Charlesworth et al. 1993b). The models of Lande (1994) and Lynch et al. (1995a, 1995b) differed from that of Charlesworth et al. (1993b) in acknowledging that individuals have a limited reproductive capacity and that the population growth rate
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may be limited by mean individual fitness. Further, Lande (1994) found that incorporation of variability in mutation effects reduced the mean extinction times by one or more orders of magnitude. Lande (1994) predicted that the accumulation of mildly deleterious alleles in finite populations poses a far greater risk to population survival than demographic stochasticity and is comparable to threats from environmental stochasticity and random catastrophes. The times until extinction under the Lande (1994) and Lynch et al. (1995a, 1995b) models are very long when compared to current captive propagation programs. Low-fecundity species restricted to finite population sizes, such as birds and mammals, are most at risk from mutation accumulation (Lynch et al. 1995b). For endangered species such as the black rhinoceros (Diceros bicornis), giant panda (Ailuropoda melanoleuca), and tiger (Panthera tigris), the minimum time till extinction for a population of Ne 5 20 represents more than 750 years under Lande’s model. It is questionable that any population is likely to persist at a stable size of Ne 5 20 for over 750 years. It is far more likely that such small populations will either perish due to other threats or grow to a larger population size. Further, cryopreservation technologies may increase the generation interval in the future, thus reducing the number of populations likely to be threatened by mutation accumulation (Soulé et al. 1986; Soulé 1989; Benford 1992; Moore et al. 1992). Typical time frames of concern for captive propagation programs are 100–200 years. The aim of current captive breeding programs, as set out by the Conservation Breeding Specialist Group of the IUCN, is for management of captive populations to maintain 90% of genetic variation for 100 years (Seal et al. 1993; Foose et al. 1995). For the low fecundity species most susceptible to mutation accumulation and of main conservation concern, this represents 25 generations or less. The Lande (1994) and Lynch et al. (1995a, 1995b) theory is expressed as mean time to population extinction. However, Lynch et al. (1995a) predicted that “. . . . they (small populations) will have a high likelihood of experiencing substantial mutational degradation within one or two centuries, the usual time frame within which most conservation policies focus.” The occurence of mutation accumulation can be detected much earlier than the extinction point through measurement of genetic load. The genetic load can be measured either directly by measuring the fitness of chromosome homozygotes or by using deliberate inbreeding to measure fitness loss. Any excess in genetic load observed over that of the base population is attributable to an increase in the genetic load through mutation accumulation. The aim of this study was to determine whether mutation accumulation poses a threat to finite sexual populations over the time span of conservation concern. We assessed whether deleterious alleles accumulated more
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rapidly in smaller than in larger populations and whether captive finite populations exhibited greater genetic load than wild populations. Three measures of genetic load were made in different sized populations of Drosophila maintained in captivity for 45 or 50 generations under benign conditions. In none of our three tests did we find evidence of mutation accumulation.
Methods Foundation and Maintenance of Lines The base population (T92) was founded from 272 impregnated females collected in February 1992 from Tyrrell’s Winery, Polkobin, in the Hunter Valley region of New South Wales, Australia. Twenty-three experimental lines were founded from T92 after two generations in captivity and maintained as pedigreed, random mating populations with effective sizes of 25 (8 replicates), 50 (6), 100 (4), 250 (3), and 500 (2) for 50 generations using single pair matings in 100 3 25 mm glass vials on PS medium (Frankham et al. 1988) at 258C. Each pair contributed only one male and one female offspring to the next generation. At generation 45 a second set of lines was generated by pooling all replicate lines of the same Ne, to ensure that any alleles fixed in individual replicates would be polymorphic and so contribute to measures of genetic load. These pooled populations (referred to as 25P, 50P, 100P, 250P, and 500P) were maintained with a population size of 750 in 15 bottles, each containing 25 pairs of parents on PS medium. They were maintained for 10 and 25 generations prior to the measurement of mutation load under benign and competitive conditions respectively to allow time for recombination and to fit with availability of people to perform the experiments. New samples captured from the wild Tyrrell’s population were used as controls for the measurement of genetic load. This wild population has exhibited stable allozyme frequencies from 1970 to the present (Franklin 1981; Frankham & Loebel 1992) and reproductive fitness estimates have been relatively stable over the 1992–1995 period (L.M.W., M.E.M., D.A.B. & R.F. unpublished data). The wild control population for the benign fitness experiment (T94) was founded from 76 impregnated females and was maintained in captivity for six generations prior to testing. The wild control population for the competitive fitness experiment (T95) was founded from 415 impregnated females and was maintained in captivity for four generations. The T92 base population was maintained under relatively competitive conditions (25 pairs/bottle) for 35 and 52 generations prior to the fitness tests under benign captive and competitive conditions respectively. The T92, T94, and T95 were maintained in 20 3 600 mL bottles with population sizes of approximately 1000.
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Measuring Genetic Load The genetic load was measured as the fitness differential between inbred and non-inbred lines derived from each of the pooled populations and controls. Samples from the pooled populations and base and wild populations were deliberately inbred for three generations using fullsib matings. The difference between the fitness of the inbred and contemporary non-inbred controls of each of the populations was assessed when the inbreeding coefficient (F ) of the inbred parent flies was F 5 0.5 and their offspring F 5 0.59. Four measures of genetic load were made for each population under both benign and competitive conditions. For measurement of reproductive fitness under benign conditions, four sets of approximately 12 replicate single virgin pairs of flies were set-up for each of the inbred and non-inbred lines of each of the pooled populations plus T92 and T94. Pairs were kept in vials containing PS medium at 258C. Parents were removed after allowing 70 hours for egg laying. The number of offspring emerging was recorded until day 16. The genetic load was calculated as the mean productivity of noninbreds minus inbreds. Fitness under crowded competitive conditions was measured using the competitive index ( Jungen & Hartl 1979). All inbred and non-inbred lines competed with the same compound chromosome stock C(2L) b; C(2R) cn bw (Lindsley & Zimm 1992). Four sets of five replicate vials were set up for each of the pooled populations, T92 and T95. Each vial consisted of five pairs of virgin experimental flies and 10 pairs of virgin compound flies in vials on PS medium at 258C. Parents were allowed 40 hours for egg laying. The numbers of wildtype and compound progeny emerging until day 18 were recorded. The competitive index was calculated as the ratio of wild-type to compound flies ( Jungen & Hartl 1979). Values used to compute the competitive index for each set were the sum of wild type and compound flies for all five replicate vials. Frequencies of Deleterious and Lethal Alleles For each of the un-pooled populations, the frequencies of deleterious and lethal alleles were estimated by isolating chromosome II (approximately 40% of the genome) using a marked balancer stock Cy/Pm : In(2LR)SM1, al2 Cy cn2 sp2/In(2LR)bwV1, ds33K dpov bwV1 (Lindsley & Zimm 1992) and making the isolated chromosomes homozygous (Muller 1928). Prior to the isolation of chromosome II, all background chromosomes of the balancer stock were replaced with those of the base population to avoid hybrid dysgenesis. Forty to fifty chromosomes were randomly sampled from each of the 23 un-pooled populations at generation 56 and the base (T92) population at generation 35. Numbers of Cy and
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wild-type offspring were counted, and chromosomes were classified according to the ratio of Cy:wild-type progeny following Choi (1978). A 2:1 ratio of curly winged (Cy) to wild-type offspring is expected from each test cross in the absence of detrimental mutations. Test crosses with ,1% wild-type offspring were classified as lethal carrying chromosomes, and those with 1–26.7% wild-type offspring as deleterious. Data Analyses Regressions of loss of fitness due to genetic load on Ne were computed. The genetic loads of the finite captive populations and the wild outbred population were compared using Kruskal-Wallis tests. Infertile matings were excluded from these analyses. Contingency x2 analyses of independence were used to test relationships between the number of infertile matings and Ne in both inbred and non-inbred populations. The x2 tests were used to assess differences in the number of infertile matings in inbred and non-inbred lines for each Ne. For statistical analyses of competitive index data, the genetic load was ranked in ascending order within each of the four sets because competitive indices are ratios and are not normally distributed. Frequencies of lethal allele-carrying chromosome II were estimated by Poisson correcting observed frequencies using equation 1 (Malpica & Briscoe 1975): corrected frequency = – log e ( 1 – Q ) ,
Figure 1. The genetic load (difference in fitness of non-inbred and inbred lines) of populations maintained for 45 generations at effective population sizes of 25, 50, 100, 250, and 500 (replicates pooled) and their base and wild control populations. Fitness was measured under both competitive and benign conditions. The left axes express genetic load relative to that of the base population. The right axes give the absolute values of genetic load as measured under either competitive (difference in mean rank of competitive index) or benign (difference in mean number of offspring per pair) conditions. Bars represent standard errors.
(1)
where Q is the observed frequency of lethal carrying chromosomes. Poisson correction was necessary to account for the occurrence of more than one mutation on a single chromosome. Regressions of the frequency of both deleterious and lethal alleles on Ne were computed. Tests of significance were computed using onetailed tests when predictions were directional. All statistical analyses were performed using MINITAB, Version 8 (Ryan et al. 1994).
Table 1. Regressions of measures of the genetic load on effective population size ( Ne ) for populations of Drosophila melanogaster maintained at sizes of 25–500 for 45–50 generations. Environment a Benign Competitive
Intercept
pb
Regression coefficient
p
41.26 6 5.06 2.39 6 0.44
,0.001 ,0.001
0.06 6 0.02 0.003 6 0.002
0.99c 0.96b
0.17 6 0.03 0.19 6 0.04
,0.001 ,0.001
Chromosome II d Deleterious Lethal
0.00019 6 0.00018 0.00018 6 0.00024
0.85c 0.45b
a Regressions of the loss of fitness due to the genetic load (outbred–inbred) on Ne for experiments conducted under benign and competitive environmental conditions. b Probability based on two-tailed t test. c Probability based on one-tailed t test of the hypothesis that small populations will have greater genetic load. d Regressions of the frequencies of deleterious and lethal allele-carrying chromosome IIs on Ne .
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Figure 2. The frequency of deleterious and lethal carrying second chromosomes in populations of Drosophila maintained for 50 generations under benign conditions at effective population sizes of 25, 50, 100, 250, and 500 and their base population. Bars represent standard errors.
Results and Discussion No clear directional trend of smaller populations suffering greater genetic loads than larger populations was found under either benign or competitive conditions (Fig. 1, Table 1). Further, the genetic load of the finite captive populations was not significantly different from that of the wild population under benign (mean 5 51.6 versus 44.0; Kruskal-Wallis H 5 0.09, df 5 1, p 5 0.77) or competitive conditions (mean rank 5 3.55 versus 4.5; H 5 0.79, df 5 1, p 5 0.37). Under benign conditions the number of infertile matings was independent of Ne in both inbred (x2 5 7.315, df 5 6, p 5 0.708) and noninbred lines (x2 5 0.515, df 5 6, p 5 0.998). Significantly more failed matings occurred in inbred than in non-inbred lines across all treatments (x2 5 17.86, df 5 6, p 5 0.007) indicating inbreeding depression. Further, there was no significant relationship between the frequency of deleterious or lethal containing chromosomes and population size (Fig. 2, Table 1). None of our experiments showed a consistent pattern of increasing mutational degradation with decreasing population size. Further, the genetic load was not greater in finite captive populations than in the wild population from which they were founded. Consequently, the accumulation of new deleterious mutations does not appear to pose a significant threat to wildlife populations with effective sizes of 25–500 over the usual time spans of conservation programs. The observed relationships between genetic load and population size were positive rather than negative as predicted by Lande (1994) and Lynch et al. (1995a, 1995b) (Figs. 1 & 2). This is consistent with the simulations of Charlesworth et al. (1992), who observed a lower genetic load in smaller than in larger simulated populations. Even at Ne 5 25, natural selection appears capable of removing deleterious mutations from the genomes of sexual populations. Drosophila is a particularly appropriate model for testing the predictions of Lande (1994) and Lynch et al.
(1995a, 1995b). The methods of maintenance ensured that the experimental populations satisfied all assumptions. Our experimental lines were maintained under benign uncrowded captive conditions that were highly favorable to the accumulation of mutations. Selection on most traits was minimal. Further, between family selection was eliminated by equalizing family sizes, effectively halving the natural selection experienced within these experimental populations. Additionally, Drosophila may be expected to exhibit higher rates of mutation accumulation than many other species as they lack crossing over in males and have only four pairs of chromosomes. If the accumulation of new deleterious mutations is a threat to small populations over time scales relevant to conservation programs, it should have been exhibited by our experimental lines of Drosophila. Why didn’t we detect an increased mutation load of the type predicted by the Lande (1994) and Lynch et al. (1995a, 1995b) models? This may have been due to unrealistic assumptions in the models, to unrealistic parameters being used, or to the short duration of our experiment. Lande’s (1994) assumption of variation in mutation effects seems realistic (Gregory 1965; Mukai et al. 1972; Edwards et al. 1987; Mackay 1990; Mackay et al. 1992; Santiago et al. 1992; López & López-Fanjul 1993; Keightley 1994). However, data on the actual distribution of mutational effects for fitness characters is limited, especially for mutations of small effect. It is possible that the actual distribution of mutation effects does not exhibit a large enough tail of weakly selected mutations for their fixation to substantially increase the genetic load in sexual populations. The parameters used by Lande (1994) and Lynch et al. (1995a, 1995b) are based on limited data and are dependent on the environment under which they were measured (Kondrashov & Houle 1994). Our results were not affected by the environment because genetic load was measured under both benign and competitive environmental conditions and the two measures were highly correlated (r 5 0.91).
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The Lande (1994) and Lynch et al. (1995a, 1995b) models do not include the effects of beneficial mutations, and so overestimate extinction risk. The occurrence of advantageous mutations has been documented in bacteria, fungi, and Drosophila (Ayala 1969; Lenski et al. 1991; Wilkes & Adams 1992; Cohan et al. 1994; Lenski & Travisano 1994; Butcher 1995; Moran 1996). Further, mutator strains of bacteria have shown a selective advantage over those with a normal mutation rate (Gibson et al. 1970; Cox & Gibson 1974; Painter 1975; Chao & Cox 1983). The theories of Lande (1994) and Lynch et al. (1995a, 1995b) are based on times to extinction as opposed to genetic load as we measured. Under these models, our experiments were too short to detect increased population extinctions. However as substantial mutational degradation can be expected within the first one or two centuries (Lynch et al. 1995a), the time frame of our experiment was sufficient to detect the effects of mutation accumulation in accordance with the models. Although the periods in stock prior to the measurement of genetic load reduced the effective number of generations, this amounted to only six generations for the chromosome II measurements. This experiment yielded similar conclusions to the determinations from the pooled populations. Conservation Implications Our results indicate that the accumulation of mildly deleterious mutations does not pose a significant threat to finite sexual populations over 45–50 generations. This number of generations is consistent with the 100–200 year time frames of most wildlife conservation programs. If greater than 45 generations are required for the accumulation of mildly deleterious alleles to reach detectable levels, it is doubtful whether it constitutes a significant threat to most wildlife populations. Within this time frame, conservation efforts will have either succeeded in enlarging the population to a size where accumulation of mildly deleterious alleles is no longer a threat or the population will have succumbed to another of the extinction processes. If the current situation of a lack of space for captive maintenance and the degradation of potential reintroduction sites continues, populations may be restricted to finite population sizes indefinitely. Further, species with shorter generation times, such as invertebrates, are now being encompassed by the captive breeding community. The use of cryopreservation is likely to become routine in the next century and greatly reduce the effects of mutation accumulation within susceptible populations. The only populations likely to have been restricted to small, stable population sizes for the periods of time consistent with the theories of Lande (1994) and Lynch et al. (1995a, 1995b) are endemic island populations. Even these populations are now unlikely to remain safe
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from anthropogenic factors for the timespans required by the Lande (1994) and Lynch et al. (1995a, 1995b) models. Mutation accumulation does not appear to represent a major threat in wildlife conservation programs.
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Conservation Biology Volume 11, No. 5, October 1997
