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Copyright 0 1995 by the Genetics Society of America

Genetic Adaptation to Captivity and Inbreeding Depression in Small Laboratory Populations of Drosophila melanogaster

B. D. H. Latter and J. C. Mulley'

School of Biolopcal Sciences and Faculty of Agriculture, University of Sydney, Australia Manuscript received February 23, 1994

Accepted for publication September 19, 1994

ABSTRACT The rate of adaptation to a competitive laboratory environment and the associated inbreeding depres-

sion in measures of reproductive fitness have been observed in populations of Drosophila melanogaster with mean effective breeding size of the order of 50 individuals. Two large wild-derived populations and a long-established laboratory cage population were used as base stocks, from which subpopulations were extracted and slowly inbred under crowded conditions over a period of 210 generations. Comparisons have been made of the competitive ability and reproductive fitness of these subpopulations, the panmictic populations produced from them by hybridization and random mating and the wild- or cage-base populations from which they were derived. After an average of -180 generations in the laboratory, the wildderived panmictic populations exceeded the resampled natural populations by 75% in fitness under competitive conditions. The cagederived panmictic population, after a total of 17 years in the laboratory, showed a 90% superiority in competitive ability over the corresponding wild population. In the inbred lines derived from the wild-base stocks, the average rate of adaptation was estimated to be 0.33 2 0.06% per generation. However, the gain in competitive ability was more than offset by inbreeding depression at an initial rate of -2% per generation. The effects of both adaptation and inbreeding on reproductive ability in a noncompetitive environment were found to be minor by comparison. The maintenance of captive populations under noncompetitive conditions can therefore be expected to minimize adaptive changes due to natural selection in the changed environment.

T HE need for minimizing adaptation to a captive environment has been stressed by a number of

authors interested in the conservation of endangered species (FRANKHAM et al. 1986; LANDE and BAR- ROWCLOUGH 1987; HEDRICK 1992) and by those involved in the introduction of insects as biological control agents (MYERS and SMATH 1980). There is ample evidence that adaptation may occur in large wild-derived laboratory populations of Drosophila, but little information is available on the behavior of small populations introduced from the wild that would provide a model for conservation biologists dealing with a comparable situation.

DOBZHANSKY and SPASSKY (1947) studied the effects of natural selection in strains of Drosophila pseudoobscura rendered homozygous for individual second or fourth chromosomes and maintained under moderately crowded conditions over a period of 50 generations, showing dramatic changes in viability, rate of emergence and the phenotypic expression of recessive mutants.

AYALA (1965) reported the progressive adaptation of

Corresponding author: B. D. H. Latter, 43 Camerons Rd., McLeans Ridges, Lismore, N.S.W. 2480, Australia.

' Present address: Department of Cytogenetics and Molecular Genetics, Women's and Children's Hospital, North Adelaide 5006, Australia.

Genetics 139: 255-266 (January, 1995)

a New Guinean and an Australian strain of Drosophila serrata and their hybrid to the conditions of intense crowding in laboratory populations maintained by the serial transfer technique. The hybrid populations increased in size from -2200 to 3200 individuals over periods of -11 generations at 19" or 18 generations at 25", primarily because of increased longevity of the adult flies. Later experiments with the same populations showed the resultant strains to have higher total size and productivity than the original laboratory stocks maintained in mass culture (AYALA 1968).

BRISCOE et al. (1992) have examined the rate of loss of genetic variation and adaptation to captivity in laboratory populations of Drosophila melunogaster maintained at sizes in excess of 1000 adult individuals per generation. The Kaduna, Pacific and Canberra cage populations, which had been kept in the laboratory for 23, 18 and 14 years, respectively, were found to have levels of heterozygosity approximately half those in five other large laboratory populations aged 0.5-3 years. Rapid loss of genetic variability associated with a threefold increase in competitive ability was also reported in the Armidale population maintained at Macquarie Univer- sity for approximately 3 years.

FRANKHAM and LOEBEL (1992) compared the competitive ability of a large population of D. melanogaster from the Hunter River Valley of New South Wales that

256 B. D. H. Latter and J. C. Mulley

had been maintained in captivity for 1 year with that of a recently caught wild population from the same locality. A serial transfer system similar to that used by AYALA (1965) was used to maintain the captive population for the first 8 mo in the laboratory environment. The competitive index for the captive population was twice that of the wild population, and adaptation to the laboratory environment was estimated to have occurred in approximately eight generations.

These reports dealing with the adaptation of wild populations of Drosophila to a laboratory environment involve actual population sizes ranging from 1000 to >3000 adult individuals. Little information is available about the rate of adaptation to captive environments and the associated inbreeding depression in reproductive fitness for small populations inbred at a rate of one or a few percent per generation. Experience with domesticated animals would suggest that this rate of inbreeding can be tolerated in captive populations (FRANKEL and SOULE 1981), provided that selection for both male and female fertility is routinely practiced as is the case in most animal breeding programs.

The study to be reported in this paper was initiated to measure the rate of adaptation to a competitive laboratory environment and the associated inbreeding depression in reproductive fitness in populations of D. melanogaster with mean effective breeding size of the order of 50 individuals. Two large wild-derived populations and a long-established laboratory cage population were used as base stocks from which subpopulations were extracted and slowly inbred under crowded conditions over a period of 210 generations. Comparisons have been made of the competitive ability and reproductive fitness of these subpopulations, the panmictic populations produced from them by hybridization and random mating and the base populations from which they were derived.

MATElUALS AND METHODS

Origin of experimental populations: The populations involved in this study have a complex history that is illustrated in broad outline in Figure 1. A wild population was initially sampled to set up a large bottle or cage laboratory population from which isolated slowly inbred subpopulations were derived, followed by intermixing of the inbred lines to form a panmictic population. The original wild population was subse- quently resampled for comparison with the resultant inbred and panmictic laboratory populations.

Canberra populations: The Canberra wild population was first introduced to the laboratory in December 1959 using >lo0 females trapped in Canberra, Australian Capital Terri- tory (LATTER 1964). It was maintained as a 20-bottle population with periodic intermixing until March 1964 and thereafter as the Canberra cage population in the Department of Animal Husbandry, University of Sydney with an estimated average size of 4000 adults (SHERIDAN et al. 1968).

In June 1970 a large sample of adults from the Canberra cage population was used to form a new 20-bottle population,

Wild

1

0000000

Population

FIGURE 1 .-Origin of the experimental populations. A sample from a wild population has been maintained in the laboratory as either a multiple-bottle population with periodic intermixing or as a large cage population. Derived subpopulations undergoing slow inbreeding and adaptation to the laboratory environment have finally been intermixed to form a large panmictic population.

from which 20 isolated subpopulations were initiated in May 1971 and maintained by synchronous mass transfer of parents using a single half-pint bottle for each subpopulation without reserves. This set of Canberra inbred lines is designated CI( n) , where n denotes the number of generations of inbreeding.

A panmictic 10-bottle population was formed by intermixing the 18 surviving subpopulations in June 1974 after 80 generations of slow inbreeding. This Canberra panmictic population is denoted by CP(80,n) to indicate its formation from the CI(80) subpopulations, followed by n generations of random mating.

Adults were again trapped from the Canberra wild population in December 1975 to establish a new 10-bottle laboratory population designated CW(n), where n denotes the number of generations of random mating since introduction to the laboratory. In June 1976 the Canberra cage population was resampled to establish a 10-bottle population designated CC( n) , where n denotes the number of generations of subse- quent random mating.

Letona populations: The Letona wild population w d S first sampled in January 1971 by Dr. B. L. SHELDON in the grounds of the Letona fruit cannery, located in Leeton in the Murrum- bidgee Irrigation Area of New South Wales. The population was initiated with over 100 wild females and maintained as a 20-bottle population with periodic intermixing. A set of 20 subpopulations designated LI( n) was initiated from this population in May 1971 and maintained in the same manner as the Canberra subpopulations.

After 120 generations of slow inbreeding, a 10-bottle panmictic population designated LP(120,n) was formed by intermixing the 16 surviving subpopulations. The Letona cannery

Genetic Adaptation and Inbreeding 257

wild population was resampled in February 1977 to set up a new 10-bottle laboratory population designated LW( n) .

Tyrrells populations: A Tyrrells wild population was first established in the laboratory with a sample of >lo0 females collected by Dr. I. R. FRANKLIN in March 1971 from the cellars of Tyrrells winery, located in the Hunter River Valley of New South Wales. A set of 20 isolated subpopulations designated TI(n) was initiated in May 1971 and maintained in the same manner as the Canberra and Letona subpopulations.

After 120 generations of slow inbreeding and again after 171 generations, panmictic 10-bottle populations denoted TP(120,n) and TP(171,n) were formed by intermixing the 16 surviving subpopulations. A second sample of over 100 wild females was taken from the Tyrrells winery in March 1978 and maintained in the laboratory as a 10-bottle population with periodic intermixing. This resampled Tyrrells wild population is designated TW(n), where n denotes the number of generations since its initiation in the laboratory.

Mutant competitor stocks: The principal stock used in the competition tests reported in this paper is Canysc, formed by 10 generations of backcrossing of the sex-linked markers y and sc into the Canberra cage population. Canysc was maintained thereafter as a 10-bottle population with periodic intermixing, additional bottles being set up as required for competition tests. Standard CyL/Pm and y s m laboratory stocks have also been used as competitors in one series of tests, their numbers being built up as required.

Maintenance of laboratory stocks: Wild-type and mutant stocks were routinely maintained at 25 t 2" in half-pint milk bottles, each containing -70 ml of yeast-fortified medium, containing 15% compressed baker's yeast, 10% treacle, 8% wheat semolina, 0.8% agar and 0.5% mold inhibitor (Ni- pagin) by weight boiled for 20 min in distilled water. After cooling the bottles were seeded with a concentrated suspension of live yeast and allowed to dry before use.

Parents were transferred fortnightly to new bottles without etherization and removed after three days. The new generation of adults was allowed to mate and feed within the old culture bottles, normally stored at 19 -+ 2" for the last three days before transfer. The number of parents per bottle was variable and regulated only by general inspection. Periodic counts of the numbers of male and female parents removed from the stock bottles of the slowly inbred lines showed the harmonic mean population size per bottle to be 66.7 -+ 2.2, with a range from 10 to 191.

Multiple bottle populations were intermixed at roughly 10- generation intervals and two or three generations before use in competition tests. Adults from the bottles were mixed in a single container and transferred to new bottles after etherization. The rate of inbreeding in 20- and 10-bottle populations is estimated to be no more than 0.05 and 0.196, respectively, based on an effective population size of -50 per bottle (UT-

Tests of competitive ability: Competitive ability has been assessed by the use of singlegeneration tests similar to the female test of LATTER and ROBERTSON (1962), replicated over a number of generations to allow estimates to be made of the repeatability of the estimate of the competitive index, and the detection of possible time trends. Wild-type and mutant individuals for use in the competition tests were raised in separateculture bottles, each initiated with 20 pairs of parents (day 1) removed from the relevant laboratory stock bottles after egg laying. The parents were removed on day 4, and the offspring were either transferred to new bottles on day 12 (fullfeeding) or retained in the old culture bottles stored at 19 ? 2" from day 12 (restricted feeding) before their use in Competition cultures. After mixing of flies from the storage

TER et Ul. 1995).

bottles, competing sets of 25:25 wild-type:mutant females were set up in new half-pint bottles on day 15 to lay for three days at 25 ? 2" in the absence of males. The competing sets of females were allowed to recover fully after etherization before transfer to the competition bottles. The offspring were collected over a 10-day period, sorted and then either weighed, counted or both.

Measurement of noncompetitive fitness: Parents were raised in the same manner as for the competition tests with full feeding during maturation. On day 15 individual nonvir- gin females were set up in 4 X 1 in. vials with -7 ml of medium to lay for eight days at 25 ? 2" in the presence of a single male. Offspring were counted or weighed over an 8- day period of emergence.

Electrophoresis: Adult flies were homogenized in buffer and sucrose, the suspension was centrifuged and the superna- tant was subjected to electrophoresis on density gradient poly- acrylamide (Gradipore) or horizontal starch gels. The 20- enzyme systems successfully scored were acid phosphatase, alcohol dehydrogenase, aldehyde oxidase, esterase-6, esterase- C, esterase-E, a-glycerophosphate dehydrogenase, hexokinase-1, hexokinase-2, hexokinase-3, isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, mannose phosphate isomerase, octanol dehydrogenase, peptidase-I, peptidase-2, phosphoglucomutase, 6phosphogluconate dehydrogenase and phosphoglucose isomerase. The staining procedures were those of BARKER and MUI.I.EY (1976), except for acid phosphatase (AYU et al. 1972), mannose phosphate isomerase (NICHOLS et al. 1973) and peptidase (PRAKASH 1973).

The Tyrrells wild and Canberra cage samples were assayed immediately after collection, and the Canberra and Letona wild samples were assayed after - 15 generations in the laboratory. The Canberra, Letona and Tyrrells panmictic populations were assayed after separation from their parental populations for -135, 165 and 170 generations, respectively, with estimated inbreeding coefficients of -0.07-0.08. A total of 40 individuals from each population was routinely scored for each enzyme system.

Statistical analyses: The competition tests in this study involve counts or weights of wild-type and mutant offspring, produced in a number of replicate singlegeneration cultures, repeated over a period of generations. Analyses of variance have routinely been carried out on the total number or weight of offspring produced by the replicates within generations, expressed as the proportion of wild-type offspring or biomass. Both arcsin (dp) and In [ p / (1 - p ) ] transformations have been used routinely for comparison with analyses of variance of the proportions p . Ratios of wild-type to mutant body weight or emergence rate have been analysed with and without a logarithmic transformation. In no instance have the conclu- sions from the analyses of variance been appreciably affected by the transformation used.

Competitive indices have been calculated as CI = mi,/ (1 - mfi) with an approximate SE given by

SE(C1) = SE(mp)/(l - m!,)', (1)

where mp denotes the mean proportion over generations. The SE of these and other ratios are presented as a guide to the level of accuracy involved, but it should be appreciated that confidence intervals around ratios may be markedly asymmet- rical and that comparisons of ratios (R) are best made after transformation to a logarithmic scale, using the approximate relationship

SE(1n R) = SE(R)/R (2)

Measures of genetic distance: The genetic distance be-


tween pairs of populations based on the observed allozyme frequencies has been calculated in two different ways, using Equations 4 and 5 given below. In the absence of natural selection, 4* can be interpreted as the extent of divergence due primarily to the balance between genetic drift and migration and is closely related to the inbreeding coefficient (LAT- TER 1973a,b; L A l T E R and SVED 1981). Nei's genetic distance D is a measure of the rate of mutational divergence between populations, measured on a scale from zero to infinity (NEI 1972).

For two populations PI and R2 with alleles Ai at frequencies p i l and pi2, respectively, 4* is given by the equation

4* = 1; (pi1 - p i2 )*I / [ l - x p i , p i * I t (3)

= 1 - H,/H,, (4)

where

H, = 1 - ;(x + p:*) is the mean level of heterozygosity within populations, and

H h = 1 - E p i l p i 2

is that predicted in the hybrid population PI X R2 (LATTER 1973a). Allozyme frequency data from different loci have been combined by the substitution of mean values of H, and Hb in Equation 4.

The value of D is given by the equation

D = -In Z, (5)

where

1 = [E p i 1 p i 2 1 / [E p:, p;1 (6)

data from different loci being combined by using arithmetic means of C p i l p i 2 , Z p:l and Z p l over all loci (NEI 1972).

RESULTS

Measurements of competitive ability: Competition between Canberra poplhtions and CyL/Pm, ysm: Tests of the performance of Canberra wild and Canberra panmictic populations in competition with the CyL/Pm or y s m stocks involved the use of wild-type and mutant inseminated females after full feeding and extended over a period of 11 consecutive generations with an average of nine replications per test. The Canberra wild population CW(13) had been maintained in the laboratory for an average of 13 generations, and the panmictic population CP(80,53) was formed by intermixing the Canberra inbred lines after a period of 80 generations of slow inbreeding, followed by an average of 53 generations of mass mating. The estimated inbreeding coefficient of the panmictic population was 0.07. Statistical tests were based on analyses of variance of single generation means over the ll-generation interval.

Figure 2 shows the mean competitive indices to be very similar for the tests using CyL/Pm and yscw as competitor, with a clear-cut superiority in both cases of the panmictic population over the wild strain based on 2- days emergence, the mean relative competitive index being 1.90 f 0.37**. It was obvious that the wild popula-

R A T

I 0

12T

Wild Panmictic c

Wild Panmictic

Ea CvuPm YSCW

FIGURE 2.-The ratio of wild-type to mutant offspring number in cultures involving Canberra wild CW( 13) or Canberra panmictic CP(80,53) in competition with CyL/Pmor y s m . The difference in competitive index between the wild and panmictic populations after 2 days emergence (early) is highly significant.

tion was characterized by a high frequency of pupation on the surface of the medium by comparison with the panmictic population. The difference between the panmictic and wild populations based on total emergence was not significant, with a mean relative competitive index of 1.16 f 0.16. No significant trends were detected over the l l generations of testing.

It can be seen from Figure 2 that the CyL/Pm and y s m females produced far fewer offspring than the wild- type females, with ratios of wild-type to mutant in excess of 5:1, indicating that the mutant stocks were relatively poor competitors. It was therefore decided to use the more vigorous Canysc stock as competitor in subse- quent tests of competitive ability.

Competition between Canberra popllutions and Canysc: After a further 24 generations of mass mating, the

competitive ability of Canberra wild and Canberra panmictic was compared with that of the Canberra inbred lines CI(157) and a laboratory 10-bottle population CC(24) derived from the Canberra cage population 24 generations earlier. The tests were carried out over a period of six generations with 10 replicate cultures per generation. Five replicates involved mating and full feeding of adult females in new bottles with live yeast before egg laying in the competition cultures, and the remaining five replicates used wild-type females that had mated and fed under restricted conditions in the parental culture bottles. The competing Canysc females were all mated and fully fed in new bottles with live yeast.

Genetic Adaptation and Inbreeding

TABLE 1

Competitive indices of Canberra inbred (CI), cage (CC) and panmictic (CP) populations in competition with Canysc, expressed relative to the Canberra wild population CW(37)

Competitive index relative to wild"

Nutrition Daysh CI( 157) CC (24) CP(80,77)

Restricted 2 0.92 t 0.27 1.49 2 0.39 2.20 2 0.54**

Full 2 0.69 2 0.16 1.63 2 0.38* 1.85 t 0.43* 10 0.77 2 0.15 1.17 2 0.20 1.28 2 0.22

10 0.50 2 0.06*** 0.70 2 0.08** 0.68 2 0.08**

C I ( n ) denotes lines slowly inbred for n generations; CC(n), a large population extracted from the cage and mass mated for n generations; CP( m,n), a large panmictic population formed from the CI( m) lines and mass mated for n generations; and CW(n), a wild population mass mated in the laboratory for n generations.

" Based on numbers of offspring. Number of days of emergence.

Significantly different from the wild population at *P = 0.05, **P = 0.01 and ***P = 0.001.

259

The data of Table 1 show that the roughly twofold superiority in 2day emergence of Canberra panmictic over Canberra wild had been maintained since the tests with CyIJPm and y s m were carried out. However, the competitive indices based on total emergence under the full feeding regime had changed appreciably, the wild population having improved in competitive ability by a factor of 1.71 ? 0.30** relative to Canberra panmictic over the 24-generation period. This superiority of the wild population was not evident under the restricted feeding regime.

The estimates of competitive ability for the Canberra panmictic and cage populations in Table 1 do not differ significantly on either the restricted or full-feeding regimes. However, the panmictic population CP(80,77) had an estimated inbreeding coefficient of 0.10 compared with only 0.02 for the cagederived population CC(24), and this difference in inbreeding is expected to have reduced the competitive index of the panmictic population for early emergence by roughly 15% (LAT- TER et al. 1995). It is therefore possible that some of the superiority of Canberra panmictic over the resampled wild population has been caused by natural selection in the slowly inbred lines, though a major part of the increase in competitive ability must have occurred in the Canberra cage base population.

Compdition between Ixtona Populations and Canysc: The Letona panmictic population LP was formed by the intermixing of all 16 surviving Letona inbred lines after 120 generations of inbreeding. After 40 generations of random mating, the competitive ability of the panmictic population was compared with that of the parental inbred lines and a Letona wild population that had been introduced to the laboratory 10 generations earlier. The estimated inbreeding coefficient of the panmictic population was -0.07. The populations were tested against Canysc over six consecutive generations, using both restricted and full-feeding regimes each with six replicate competition cultures per generation.

The competitive indices derived from the mean proportions of wild-type individuals emerging from the competition cultures are shown in Figure 3 separately for the two parental feeding regimes and for early and total emergence of offspring. The competitive ability of the inbred and panmictic populations relative to the Letona wild population is summarized in Table 2. The inbred lines were not significantly different from the wild population under the regime of restricted parental nutrition but were significantly less competitive under the full-feeding regime. The panmictic population, on the other hand, was significantly superior in competitive ability to the wild population under both regimes. It is apparent that the wild population was especially

7

R A T I 0

0.5

0.0 Restricted

+

Total

Restricted 4.

Total

Full Full IC

Inbred Wild N Pan

FIGURE 3.-The ratio of wild-type to mutant offspring number in Letona inbred, wild and panmictic populations competing with Canysc. Early refers to offspring produced within 2 days of emergence. Restricted and full refer to the feeding regimes used for the competing Letona adult individuals.

260 B. D. H. Latter and J. G. Mulley

TABLE 2 Competitive indices for Letona inbred (LI) and panmictic (LP) populations in competition with Canysc, expressed

relative to the Letona wild population LW( 13)

Competitive index relative to wild"

Nutrition Daysb LI ( 163) LP(120,43)

Restricted 2 1.28 5 0.54 5.31 ? 1.84*** 10 0.90 ? 0.14 2.01 2 0.26***

Full 2 0.70 ? 0.12* 1.50 t 0.23* 10 0.71 ? 0.05*** 1.81 2 0.12***

LI( n) denotes lines slowly inbred for n generations; LP ( m, n) , a large panmictic population formed from the LI ( m) lines and mass mated for n generations; and LW(n), a wild population mass mated in the laboratory for n generations.

Based on numbers of offspring. 'Number of days of emergence. Significantly different from the wild population at * P =

0.05 and ***P = 0.001.

disadvantaged when parental feeding was restricted and when the competitive index was based on the first 2 days of emergence, presumably because of a longer delay in egg laying in the wild females.

The relative emergence rate of the Letona panmictic population, expressed as the percentage of total wild- type emergence occurring by the second day relative to the same measure for Canysc, was 2.72 5 0.70** times that of the Letona wild population in the competition cultures with restricted parental feeding. The corresponding ratio for the full-feeding regime was 0.87 % 0.20.

Competition between Tyrrells pqbulations and Canysc: Ta- ble 3 summarizes the performance of Tyrrells inbred,

wild and panmictic populations in competition with Ca- nysc, in cultures initiated with inseminated wild-type and mutant females after full feeding. The Tyrrells panmictic populations TP(120,67) and TP(171,16) were initiated by intermixing the inbred lines after, respectively, 120 and 171 generations of slow inbreeding followed by 67 and 16 generations of random mating, and they reached estimated inbreeding coefficients of -0.10 and 0.05. Emergence rate is expressed as the number of offspring emerging in the first two days, as a percentage of total emergence over a loday period. Fly weight, number of offspring and biomass are means calculated from the total offspring produced in 10 days. The least significant difference figures have been derived from analyses of variance of single-generation means over a 13-generation interval.

Despite the existence of large and highly significant differences between populations in the production of both wild-type and mutant biomass, differences in total biomass were nonsignificant. The available resources were therefore fully utilized by the competing populations, any increase in the production of wild-type biomass being at the expense of the mutant competitor. By comparison with the Tyrrells wild population, which was introduced into the laboratory three generations before the initiation of the competition tests, the inbred lines were significantly later to emerge and lower in mean body weight; they also allowed significantly greater numbers of mutant competitors to emerge (Ta- ble 3). Both panmictic populations, on the other hand, produced significantly higher numbers of wild-type individuals than the wild population, with comparable reductions in the number of mutant competitors. The

TABLE 3

Components of fitness in Tyrrells/Canysc competition cultures

Population"

Trait Genotype TI(187) TW(l0) TP(120,67) TP(171,16) LSD

Emergence rateb Wild-type 9.4 15.0 13.1 10.6 2.8 Mutant 31.9 27.4 20.1 17.5 4.3 Ratio 0.33 0.56 0.68 0.63 0.17

100-fly weight (mg)" Wild-type 71.7 79.3 80.0 79.9 2.1 Mutant 74.6 71.6 69.7 69.0 2.3 Ratio 0.96 1.11 1.15 1.16 0.04

No. of offspring' Wild-type 269 278 415 349 32 Mutant 322 238 151 157 44 Total 59 1 516 566 506 63

Biomass (mg) Wild-type 193 220 332 278 25 Mutant 240 170 105 109 32 Total 433 390 437 387 NS

TI( n) denotes lines slowly inbred for n generations; T W ( n), a wild population mass mated in the laboratory for n generations; and TP(m,n), a large panmictic population formed from the TI(m) lines and mass mated for n generations. ' Percentage of offspring produced by the second day of emergence. "Means over the total loday period of emergence.


h

Wild

1

I lnbreds 0 4 : : : : : : : : : : : ' 0 2 4 6 8 10 12

GENERATION

FIGURE 4.-The ratio of wild-type to mutant total biomass in single generation competition tests between Tyrrells populations and Canysc over a 13generation period. The panmictic populations TP(120,67) and TP(171,16) are consistently superior to the newly established wild population.

ratio of wild-type to mutant body weight was also significantly higher in the panmictic populations than in the wild.

The mean ratios of wild-type to mutant total biomass for the four Tyrrells populations are shown in Figure 4 for individual generations; each mean is based on 10 replicate cultures. The trends throughout the 13-generation period were nonsignificant for all populations, based on regression analyses of residuals after an analysis of variance of the mean proportions. Competitive indices expressed relative to the Tyrrells wild population are presented in Table 4. The panmictic populations were significantly higher than the wild population

in relative body weight and offspring number, with biomass competitive indices averaging more than twice that of the wild population.

Based on analyses of variance of the proportions of wild-type individuals or biomass produced under competition, TP(120,67) showed a significantly higher competitive index than TP(171,16) for both total offspring number and biomass, the ratio of the indices being 1.25 -+ 0.12". Similar nonsignificant differences were observed for early offspring number and biomass (Ta- ble 4). These observations suggest a rate of increase of competitive ability in the panmictic populations of 0.44 -+ 0.19% per generation over and above that in the inbred populations. This estimate has been calculated as 100[ (1.25)"51 - 11, because the two populations differ by 51 generations of random mating in a large 10-bottle population versus continued inbreeding in the small single-bottle populations. The total increase in competitive ability due to more effective natural selection in the large population TP(171,16) is then expected to be (1.0044) l6 = 1.07 ? 0.03 times the increase in the inbred subpopulations over the same 16-generation interval. Allowance for the difference of 0.05 in the inbreeding coefficients of TP(120,67) and TP(171,16) raises this ratio to only 1.11. The observed competitive indices of 2.28 and 1.98 for biomass at two days and 10 days emergence in TP(171,16) relative to TW(10) can therefore be attributed primarily to the cumulative effects of natural selection in the inbred subpopulations before hybridization.

Fitness under noncompetitive conditions: Compari- sons have been made of the Canberra inbred, wild and panmictic populations with average inbreeding coefficients of 0.54, 0.01 and 0.07, respectively, based on counts of early and total offspring from single female vial cultures. The first experiment involved 20 replicate cultures of the wild and panmictic populations and 30 replicates of the inbreds in each of six consecutive gen-

TABLE 4

Components of early and total biomass for Tyrrells inbred (TI) and panmictic ( T P ) populations in competition with Canysc, expressed relative to the Tyrrells wild population TW(l0)

Competitive index relative to wild

Days" Trait TI(187) TP(120,67) TP(171,16)

2 Emergence rate" 0.59 2 0.12** 1.22 ? 0.16 1.13 ? 0.16 100-fly weight 0.98 ? 0.02 1.06 ? 0.02** 1.06 ? 0.02** Offspring number 0.39 -C 0.06*** 2.62 ? 0.29*** 2.15 ? 0.24*** Biomass 0.37 ? 0.05*** 2.79 ? 0.31*** 2.28 ? 0.25***

Offspring number 0.72 ? 0.06*** 2.36 ? 0.21*** 1.89 ? 0.16*** Biomass 0.62 ? 0.05*** 2.45 2 0.23*** 1.98 ? 0.17***

10 1 00-fly weight 0.87 ? 0.02*** 1.04 ? 0.02" 1.05 ? 0.02**

Number of days of emergence. 'The number of wild-type offspring emerging withint 2 days as a proportion of the total number of wild-

Significantly different from the wild population at *P = 0.05, **P = 0.01 and ***P = 0.001. type offspring, relative to the same measure for the mutant competitor.


TABLE 5

Productivity of single females of Canberra inbred (CI), wild ( C W ) and panmictic (CP) populations under noncompetitive conditions

Population

Trait CI(130) CW(l0) CP( 80,50)

Percentage sterile cultures" 2.7 t 1.2 5.0 +- 2.0 1.7 +- 1.2 Emergence rate' 0.45 ? 0.013 0.46 ? 0.013 0.50 t 0.013 Offspring/fertile culture 203 -+ 3.1 200 ? 3.1 206 t 3.1

a Cultures producing <20 offspring. Proportion of offspring emerging on the first day of counting.

erations, commencing after the wild populations had been in the laboratory for seven generations. The second experiment involved 40 replicate cultures of the cage and panmictic populations in each of three generations, commencing three generations after sampling from the Canberra cage population. This comparison of the cage and panmictic populations was repeated 13 generations later, using the same cage sample maintained throughout as a 10-bottle population. The inbreeding coefficients of the cage populations were in the range 0.005-0.02.

The results of the first experiment are presented in Table 5. An average of 3% of cultures were sterile, and the mean number of offspring produced per fertile female was 203. The differences between populations were nonsignificant. The rate of emergence was slightly lower in the inbred and wild strains than in the panmictic population, but the observed differences were not statistically significant. In the second experiment, the cage and panmictic populations produced an average of 2% sterile cultures and 212 offspring per fertile culture, of which a proportion 0.43 emerged on the first day of counting. None of the differences between the two populations was significant.

Measures of genetic variation and distance: Nine of the 20 enzyme systems scored were polymorphic in one or more of the seven Australian populations involved in this study, viz. acid phosphatase, alcohol dehydrogenase, esterase-6, esterase-C, a-glycerophosphate dehydrogenase, hexokinase-3, octanol dehydrogenase, phosphoglucomutase and Gphosphogluconate dehydrogenase. The observed level of heterozygosity in the Canberra, Letona and Tyrrells wild populations did not differ significantly, averaging 0.091 ? 0.010 over the 20 allozyme systems scored (Table 6). An analysis of the variation shown by polymorphic loci showed the Can- berra cage and panmictic populations to have a significantly lower level of heterozygosity than the remaining wild and panmictic populations, with a mean of 0.054 5 0.012 over the 20 allozyme systems.

Two measures of the genetic distance between pairs of populations have been calculated, viz. +* and D, based on Equations 4 and 5. The values of the two

parameters presented in Table 6 are closely correlated, but the mean of +* is 0.084 and that of D is 0.008. This multiple of the order of 10 is shown by all pairs of populations and is indicative of the fundamental difference between the two measures. For an island model of population differentiation with migration between subpopulations, the equilibrium value of +* is deter- mined primarily by the balance between genetic drift and migration (LATTER 1973a; LATTER and SVED 1981), whereas the equilibrium value of D is a function of mutation pressure and migration rate (NEI 1972).

It is possible to estimate the contribution of sampling error to the values of +* and D when r individuals per population are scored for each locus. Genetic sampling is expected to decrease H, by a factor of [ l - (2r)" I and to leave Hb unchanged. For r = 40 the ratio Hw:Hb is therefore expected to decrease by 0.0125 and +* to increase by the same amount. The values of D are similarly expected to be increased by -0.001 because of sampling error.

The relationships among the source populations Canberra wild, Canberra cage, Letona wild and Tyrrells wild are illustrated in Figure 5 in the form of a dendrogram, derived by the unweighted pair group method of clustering based on estimates of +*. The Canberra cage population, which had been maintained in the laboratory for a total of almost 18 years, has clearly diverged from the three wild populations, the average estimated distance being +* = 0.108 with the most con- spicuous differences observed at the a-Gpdh and Pgm loci. The three wild populations show a mean divergence of +* = 0.063, which can be compared with the value of 0.075 -+ 0.010 calculated for nine species of Drosophila by LATTER (1981).

Also shown in Figure 5 is the dendrogram for the three panmictic populations derived from Canberra cage, Letona wild and Tyrrells wild after isolation in the laboratory for an average of more than 150 generations. Letona and Tyrrells panmictic show a genetic distance of +* = 0.100, which is substantially greater than the value of 0.061 for the corresponding wild populations (Table 6) . The difference is primarily due to divergence in gene frequencies at the a-Gpdh and P p loci.

Genetic Adaptation and Inbreeding

TABLE 6

Estimates of genetic distance and heterozygosity for allozyme systems in Canberra, Letona and Tyrrells wild populations and their derivatives

263

Population“ ~~

cw cc CP L” LP Tw TP

cw cc CP LW LP Tw TP Hwb

0.014 0.009 0.008 0.009 0.006 0.006 0.094

- 0.134

0.004 0.010 0.012 0.008 0.008 0.068

- 0.120 0.067

0.008 0.010 0.004 0.008 0.041

-

0.073 0.109 0.104

0.001 0.006 0.007 0.090

-

0.083 0.129 0.125 0.01 3

0.008 0.01 1 0.087

-

0.054 0.082 0.060 0.061 0.073

0.007 0.089

-

0.055 0.088 0.106 0.066 0.100 0.066

0.092 -

C#J * above, D below diagonal. “Canberra wild, Canberra cage, Canberra panmictic, Letona wild, Letona panmictic, Tyrrells wild and

hMean level of heterozygosity based on 20 allozyme systems. Tyrrells panmictic populations, respectively.

DISCUSSION

Genetic adaptation to captivity: A consistent feature of the comparisons of wild and panmictic laboratory populations is the superiority of the laboratory populations under competitive conditions when only the early emerging offspring are counted, ie., the first 10-15% of wild-type emergence (Tables 1-4, Figures 2 and 3). This adaptation to crowded conditions in laboratory cultures has occurred in three wild populations origi- nating from an urban area, a fruit processing factory and a winery, separated by distances of roughly 250- 500 km. Both the original and repeat sampling of these populations involved >lo0 adult females and was made at the same time of the year in each locality.

Figure 6 depicts the relative values of the competitive index based on the first two days of emergence of offspring from the competition cultures (Figure 2, Tables

G!E: LW

I I I I 0.15 0.10 0.05 0.00

FIGURE 5.-Genetic distances between populations (C#J*) based on estimates of allozyme frequencies at nine polymorphic loci. The source populations are Canberra wild [CW(15)],Canberracage [CC(l)],Tyrrellswild [TW(l)]and Letona wild [LW( 15)]. The derived populations are Canberra panmictic [CP(80,32)], Tyrrells panmictic [TP(120,46)] and Letona panmictic [LP(120,36)].

1,2 and 4). This measure comes closest to an assessment of the ability of the population to survive and reproduce efficiently in a competitive environment similar to that in which the inbred subpopulations were maintained in the laboratory. The index assesses the ability of a population to survive as a unit, involving male fertility, female fecundity and larval survival under conditions in which total biomass is limited by the available resources, and a premium is placed on early emergence of the adult offspring (Table 3). The ability of males to compete with unrelated males in mating is not a

c Canberra Letona Tynells

Inbred Wild ESl Pan

FIGURE 6.-The relative competitive ability of inbred, wild and panmictic populations, based on the ratio of wild-type to mutant offspring emerging within 2 days, with full feeding of the parental females. The comparisons were made after an average of 145,163 and 187 generations of slow inbreeding in the Canberra, Letona and Tyrrells populations, respectively.


component of the index. The relative merits of such single generation tests of competitive ability by comparison with alternative procedures have been discussed by HAYMER and HARTL (1982, 1983), SVED (1989) and LATTER and SVED (1994).

The Canberra, Letona and Tyrrells inbred lines in Figure 6 were the survivors of an average of 157, 163 and 187 generations of slow inbreeding without any attempt being made to accommodate slowly emerging populations or to maintain reserve cultures. During this period 23% of the subpopulations were lost, sometimes because of the transfer of only male parents when all the females perished in the sticky medium of the old culture. No line was lost by accidental destruction of the culture, and there was no advance indication of extremely low fertility in any of the lines that failed to reproduce. The estimated harmonic mean parental population size in the slowly inbred subpopulations was 66.7 t 2.2, with a range from 10 to 191 in the sample of 680 cultures counted. Evidence will be presented in a separate paper to show that the initial rate of inbreeding was 0.96 -+ 0.16% per generation, corresponding to an effective population size of -50, based on estimates of the drift variance among replicate subpopulations in allele frequencies at the Adh and Est-6 loci. However, the rate of inbreeding was not constant throughout the period of the experiment but declined with time to give an estimated inbreeding coefficient of F = 0.56 -+ 0.04 at generation 170. The corresponding panmictic populations had an average inbreeding coefficient of -0.07 at the time of testing (LATTER et al. 1995).

In a project of this sort spanning many years, it is inevitable that some immigration and intermigration must have occurred despite the extreme care taken each generation to avoid contamination. Transfer to new cultures was without etherization, so that accidental carryover of flies in the etherizer was not a possible mechanism of cross contamination. Occasional mutant phenotypes were detected at low frequency, which dis- appeared after a few generations, most of which must have been due to the occurrence of mutation rather than migration, because the mutants concerned were not kept in the laboratory. One culture was found to contain ysc individuals, presumably because of contamination from the Canysc population, and another con- tained ebony-bodied flies that could have originated from a laboratory stock. The appropriate model for these experiments must therefore include the possibil- ity of migration, but the data presented by LATTER et al. (1995), showing the frequency of chromosome in- versions in the slowly inbred lines at generation 210, indicate that the rate of migration was at an extremely low level.

The comparisons of inbred, wild and panmictic populations in Figure 6 are based on the data of Figure 2

and Tables 1, 2 and 4, using the estimates obtained for TP( 171,16) from Table 4. The Canberra, Letona and Tyrrells wild populations were tested after an average of 25, 13 and 10 generations, respectively, in the laboratory and were maintained as 10-bottle populations. The corresponding panmictic populations had been maintained as 10-bottle populations for an average of 65, 43 and 16 generations, respectively, after their formation. The mean competitive index of the Letona and Tyrrells panmictic populations in Figure 6 relative to the corresponding wild population is 1.75 t 0.18***, corresponding to an average rate of adaptation of 0.33 % 0.06% per generation over the 170-generation period involved. There is direct evidence in the case of the Tyrrells populations that this adaptation has occurred primarily in the inbred subpopulations before intermat- ing; the data of Table 4 allowed an estimate to be made of the rate of evolution in the panmictic population. The superiority in competitive index of the Canberra panmictic population over the resampled wild population is of the same magnitude as those observed in the case of Letona and Tyrrells, the mean ratio from Figure 2 and Table 1 for early emergence being 1.89 % 0.36**. However, the data in Table 1 for Canberra cage and panmictic populations suggest that a major part of this superiority evolved during the 11 years before the initiation of this project.

There is ample evidence that the adaptive changes in these laboratory populations have occurred gradu- ally, in contrast to the rapid change over a period of about eight generations reported by FRANKHAM and LOEBEL (1992). The data of Figure 4 for the Tyrrells wild population between generations 4 and 17 give an estimated nonsignificant rate of increase in competitive index for total biomass of 1.4 t 1.3% per generation relative to Canysc; the corresponding estimate for early biomass is also nonsignificant (0.2 t 1.8% per generation). The data of Figure 2 and Tables l and 2 similarly show that the Canberra and Letona wild populations were far less competitive than the corresponding panmictic populations after average periods of 13, 37 and 13 generations had elapsed after their reintroduction into the laboratory, especially with respect to the competitive index based on early emergence. The data of Table 4 also suggest that the Tyrrells panmictic populations have continued to adapt to laboratory conditions after >120 generations in the laboratory.

It appears that adaptation to the competitive conditions in laboratory cage or bottle populations has involved a number of facets of the life cycle, including earlier egg laying after restricted feeding of adults, increased pupation height, the production of higher numbers of surviving offspring and greater reduction in the emergence of competing genotypes. It should be noted that density-dependent natural selection favoring increased pupation height in D. melanogaster has been


extensively documented by MUELLER and SWEET (1986) and JOSHI and MUELLER (1993).

Computer simulation studies to be reported else- where show that the genetic basis for such changes may be comparatively simple, involving as few as 10-20 loci with alleles that have very slightly disadvantageous or neutral average effects in wild populations under natural conditions but with deleterious effects in the captive environment. It is also clear that the observed increases in competitive ability are far too large to be explained by the elimination during inbreeding of deleterious recessive genes of large effect that are initially at equilibrium frequencies. This process can at most account for increases of the order of 5-10%.

Inbreeding depression in the small populations: Comparisons of the inbred and panmictic populations

in Figure 6 show clearly that inbreeding depression in competitive ability has occurred in each of the three sets of slowly inbred populations. The mean competitive index of the inbred lines relative to the panmictic populations is 0.34 % 0.04 with estimated inbreeding coefficients of 0.56 and 0.07, respectively. This corresponds to a rate of loss of competitive ability of 2.2% per 1% increase in the inbreeding coefficient, i e . , an initial rate of inbreeding depression of -2% per generation. If allowance is made for the estimated increase in adaptation in the Letona and Tyrrells panmictic populations during the period of random mating after their formation, the estimated mean rate of inbreeding depression is only slightly changed to 1.8% per generation.

The inbreeding depression in competitive ability re- corded in this study has involved a decrease in both the number and size of adult offspring produced, a decrease in the mean rate of development and the emergence of greater numbers of mutant competitors of larger body size. Further information on the extent of inbreeding depression under competitive and noncompetitive conditions is given by LATTER et al. (1995).

Genetic distance among populations: The allozyme frequency data give an estimated mean genetic distance among the three wild populations of +* = 0.063 (Table 6, Figure 5), which is similar in magnitude to the value of0.075 % 0.010 calculated for surveys of nine Drosophila species by LATTER (1981). The Canberra cage population, on the other hand, shows a mean divergence from the wild population of 4* = 0.108 t 0.015 (Figure 5) due to its long period of isolation in the laboratory at an effective population size possibly as low as 220 adult individuals (BRISCOE et al. 1992). The Canberra panmictic population shows a similar mean divergence from the Letona and Tyrrells panmictic populations of +* = 0.1 16. The mean frequency of heterozygotes in the Canberra cage and panmictic populations has also been shown to be significantly less than that in the wild populations.

The mean divergence of the Canberra, Letona and

Tyrrells panmictic populations from their respective base populations Canberra cage, Letona wild and Tyr- rells wild, after separation for between 135 and 170 generations, was $* = 0.05 % 0.02. The expected value for this measure of genetic distance is equal to one-half the inbreeding coefficients in the derived panmictic populations, i.e., 0.03-0.04. The observed mean value of +* is therefore in reasonable agreement with expec- tation, though its SE is large.

Implications for conservation biology: These observations give some understanding of the genetic changes that may be expected in comparatively small populations maintained under crowded conditions in a novel environment, separating adaptive changes due to natural selection from those caused by the slow inbreeding involved. The implications for conservation biologists and the breeders of domesticated plants and animals are threefold. (1) Inbreeding depression in competitive ability, of the order of 2% per 1% increase in the inbreeding coefficient, may occur in outbred populations with an effective population size of N, = 50; this will correspond to an initial rate of decrease of 2% per generation. (2) A gradual increase in adaptation to the novel environment, at an estimated rate of 0.33 %

0.06% per generation in the present study, may result if the populations concerned are maintained under competitive conditions. (3) Under noncompetitive conditions the effects of both inbreeding depression and adaptation in such populations may be minor by comparison with those under competitive conditions.

The rate of inbreeding depression in competitive ability observed in these slowly inbred populations is roughly 70% that shown by the full-sib inbred lines studied by LATTER and ROBERTSON (1962), which declined by 2.9 -+ 0.2% per l % increase in F (recalculated from the data of their Table 3). However it should be borne in mind that the competitive index in the earlier study involved competitive male mating ability, and that full-sib inbred lines lost during the inbreeding process were assumed to have zero fitness. The test of competitive ability employed in the present study measures the ability of the slowly inbred populations to thrive under crowded conditions that do not involve competition from unrelated males.

The estimated rate of increase in competitive ability of 0.33 t 0.06% per generation under natural selection in a novel laboratory environment is considerably less than the 2% per generation increase in population size estimated by AYALA (1969) for control populations of D. serrata and D. birchii and the very rapid rate of increase in competitive index reported by FRANKHAM and LOEBEL (1992). It is not easy to account for the differences between these studies, because the nature of the culture medium and conditions of crowding during adaptation are not comparable. It appears likely, however, that the extremely high density and high mortality


achieved with the serial transfer system used by AYAL.A (1965, 1969) and FRANKHAM and LOEBEL (1992) is a major factor in accelerating the rate of change in the populations concerned. The important point to stress about the present project is that adaptation has taken place primarily in small populations of effective size of the order of 50, yet the rate is extremely high in evolutionary terms, leading to a doubling of competitive fitness in only 200 generations.

Dr. R. FRANKHAM and the reviewers made valuable suggestions for the improvement of earlier versions of this paper. We are grateful to Ms. D. EI.IAS and Ms. D. REID for their dedicated technical assistance in this long-term project.

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Communicating editor: T. F. C. MACKAY

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