Aquaculture, 84 (1990) 199-209 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

199

Superior Developmental Stability of Heterozygotes at Enzyme Loci in Scdmo salar L.

G. BLANCO, J.A. SANCHEZ, E. VAZQUEZ, E. GARCIA and J. RUB10

Departamento de Biologia Funcional, Area de Genktica, Universidad de Oviedo, 33071 Oviedo,

Asturias (Spain)

(Accepted 10 June 1989)

ABSTRACT

Blanco, G., Sanchez, J.A., Vazquez, E., Garcia, E. and Rubio, J., 1990. Superior developmental stability of heterozygotes at enzyme loci in Salmo salar L. Aquaculture, 84: 199-209.

The degree of heterozygosity of five enzymatic loci is related to the degree of fluctuating asym- metry of three bilateral traits in two farm stocks and in two natural populations of Salmo salar.

In all cases we verified the existence of an inverse correlation of the degree of heterozygosity with the number of asymmetrical traits per individual as well as with the total magnitude of asymmetry that the individuals present. We also detected the association at the populational levels: a higher average populational heterozygosity implies a lower total magnitude of asymmetry. The phenom- enon is general and the possibility that the correlation is due to the direct effect of a particular locus is excluded. The use of fluctuating asymmetry is proposed as an indicator of the loss of heterozygosity in salmon populations.

INTRODUCTION

Many data published in recent years relate populational or interindividual variance and the degree of asymmetry of biometrical traits with the level of heterozygosity, of populations or individuals, at enzymatic loci (see Mitton and Grant, 1984, for a review). These general data ratify the hypothesis of Lerner (1954)) according to which individuals or populations with higher genie heterozygosity possess a higher homeostasis or stability of development, shown by a lower degree of bilateral asymmetry and a lower phenotypic variance in their biometric traits (Soul&, 1979; Kat, 1982; Leary et al., 1983).

In addition to its association with developmental stability, heterozygosity is positively associated with a superior growth rate (see Mitton and Grant, 1984, for a review) and it is often associated with superior viability (Mitton and Koehn, 1975; Koehn et al., 1976; Allard et al., 1977; Zouros et al., 1983; Farris and Mitton, 1984), feeding rate (Garton, 1984; Garton et al., 1985) and fe- cundity (Koehn, cited in Mitton and Koehn, 1985 ) .

Mitton and Koehn (1985) suggest that these observations are not causally

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“00 G. BLANC0 ET AL.

unrelated, but that enzyme heterozygosity enhances physiological efficiency by decreasing the energetic cost of standard metabolism (e.g., Koehn and Shumway, 1982; Garton, 1984; Garton et al., 1985)) and that this, in turn, tends to stabilize development.

To determine the generality and the strength of this association is very im- portant in an applied and predictive context.

In this sense, there is no doubt about the importance of verifying the relation between the productive traits and enzymatic heterozygosity. As an example, Mitton and Grant (1984) state that with the association data between growth rate and heterozygosity in pine trees, they can anticipate that the cost of an electrophoretic screening of forest tree seedlings prior to restocking would be more than repaid by the enhanced growth rate of highly heterozygous individ- uals chosen for planting.

The relationship between asymmetry and heterozygosity also has applied value. In this sense, Leary et al. (1984), examining the association between heterozygosity at 42 enzyme loci and the fluctuating asymmetry of five bilat- eral meristic characters in 14 population samples of rainbow trout, cutthroat trout and brook trout, found that the more heterozygous individuals at isozyme loci had reduced fluctuating asymmetry. This relationship appears to be gen- eral among salmonid fishes. Given that fluctuating asymmetry has been as- sumed to be a reliable measure of developmental stability and to be positively associated with fitness (Sould, 1982; Soule and Cuzin-Roudy, 1982; Leary et al., 1984), these authors suggest that heterozygosity is positively correlated with fitness in these fishes.

In this work, we have tried to find out if a relationship can be established between the fluctuating asymmetry of three bilateral biometric characters and five polymorphic loci in two hatchery stocks and two natural population sam- ples of Salmo salar. The null hypothesis tested here is that there is no corre- lation between the proportion of loci that are heterozygous and the proportion of traits that are asymmetric within individuals. We have previously published data that relate higher morphological variance and/or asymmetry of five mor- phometric characters with the heterozygosity of five enzymatic loci in a stock of Atlantic salmon and where we rejected this null hypothesis (Vazquez et al., 1988). Our object here is to extend this verification using another hatchery stock and two natural populations to see if the existence of this association can be generalized in this species.

MATERIALS AND METHODS

Samples In this study we analysed samples of two natural populations and two hatch-

ery stocks of Salmo salar L. Adult fish were sampled in 1987 from the natural populations in two rivers of Asturias (northern Spain). The samples were des-

DEVELOPMENTAL STABILITY OF HETEROZYGOTES IN SALMO SALAR 201

ignated Sella and Esva according to the name of the river where they were caught. The number of adult fish studied in the Sella sample was 83 and in the Esva sample 21.

The hatchery stocks were imported as eggs from Norway (in 1986) and from Scotland (in 1987) and raised in an Asturian hatchery to be used to repopulate Asturian rivers. In both stocks one hundred 7-month-old fry were studied.

Electrophoresis Tissue samples of liver and muscle were analysed by horizontal starch gel

electrophoresis and scored for 26 enzyme loci. Details of the electrophoretic procedures, alleles and genotypic frequencies are given in Blanc0 et al. (1988) and Vazquez et al. ( 1988). In all cases, only five enzyme systems were revealed as polymorphic in the samples: Mdh-3, Idh-3, Aat-2, Sdh-1 and Sdh-2.

Morphometric characters Fluctuating asymmetry of bilateral traits was used as a measure of devel-

opmental stability because it is considered to reflect the inability of an orga- nism to develop precisely along determined pathways (Mather, 1953; Lerner, 1954; Thoday, 1958; Van Valen, 1962; Soule, 1967; Felley, 1980; Leary et al., 1984). Fluctuating asymmetry is said to occur when the difference between a character on the left and right sides of individuals is normally distributed about a mean of zero (Van Valen, 1962). Increased developmental stability is re- flected by reduced amounts of fluctuating asymmetry.

Counts of the following three bilateral meristic characters were taken on the left and right side of each fish: gill rakers on the first branchial arch, rays in the pectoral fins, and rays in the pelvic fins. These characters have been used in this type of study in the rainbow trout (Salmo gairdneri) by Leary et al. ( 1983 ) , and were chosen because they can be easily and accurately counted. Pairwise F and t-test revealed no significant differences in the variance and means of the distributions of these characters between the sexes, a fact which allows the data from different sexes to be combined for the analyses. All of the characters, therefore, exhibit fluctuating asymmetry in the populations.

In order to be able to compare directly the fishes of different ages and sizes, we chose to work with a group of meristic traits that are determined or “fixed” early in development, because it is difficult to determine what correction should be used for other morphometric measurements that increase throughout the life of the fish (Valentine and Soule, 1973; Valentine et al., 1973; Soule and Cuzin-Roudy, 1982).

For each character we calculated: - the average asymmetry (A) estimated as mean absolute value of [number on right side - number on left side] ; - the magnitude of asymmetry (D) reflecting the minimum and maximum values of deviation from bilateral symmetry.

202 G. BLANC0 ET AL.

All the enzymatic loci were analysed in each individual, as well as the bilat- eral meristic characters which allow us to establish within each sample if there is a relationship between developmental homeostasis and heterozygosity.

RESULTS AND DISCUSSION

The null hypothesis tested here is that heterozygous individuals have the same level of symmetry for each character studied as the individuals homozy- gous at the enzyme loci.

Table 1 shows the average asymmetric (A) and the magnitude of asymmetry (D ) of each character in each population for the homozygous and the different types of heterozygous individuals at enzymatic loci. The values of asymmetry were compared with a t-test corrected for unequal variances (Sokal and Rohlf, 1981) . In all the populations and for the three characters under study, we found that enzymatic homozygotes present higher levels of asymmetry. In all cases the level of asymmetry decreases inversely with the number of heterozygotic enzymatic loci.

These differences in asymmetry were significant in two of the nine cases (22%) when the homozygotic values were compared with the heterozygotic ones for a given enzymatic locus. But seven out of the twelve possible compar- isons (58% ) were significant when the homozygotic values were compared with the heterozygotic for two or more loci (Table 1 ), a fact that seems to indicate that the asymmetry of a character decreases as the number of heterozygotic loci increases. It is also interesting to point out that the magnitude of asym- metry (D) of the three characters decreases as the number of heterozygotic loci increases, a fact indicating that heterozygosis diminishes the values of asymmetry.

These data lead to rejection of the null hypothesis tested here, and are con- sistent with the alternative hypothesis based on data regarding allelic isozyme variation in several species of plants and animals that suggest that heterozy- gous individuals exhibit lower levels of fluctuating asymmetry and morphol- ogical variance than do more homozygous individuals (for reviews see Allen- dorf and Leary, 1986; Zouros and Foltz, 1987)) and the effect can be evidenced with the variation of only one locus (Mitton, 1978).

On the other hand, we also tested the hypothesis that there is no correlation between the proportion of loci that are heterozygous and the proportion of traits that are asymmetric within individuals. For this analysis the number of heterozygous loci and the number of asymmetric characters per fish both ranged from zero to three and correlated (Table 2).

The results of correlation analysis between the proportion of heterozygous loci and asymmetric characters per individual, using a t-test with an arc sine transformation for proportions (Sokal and Rohlf, 1981), are summarized in Table 2. In the two domestic populations and in one of the natural populations

DEVELOPMENTAL STABlLITY OF HETEROZYGOTES IN SALMO SALAR 203

TABLE 1

Comparisons of average asymmetry for homozygote and heterozygote classes

Sella sample

Character Horn. Het.-1 Het.-2+3 Horn. vs. Het.-1 Horn. vs. Het.-1+3

Pectoral A fin rays D

I7

Pelvic A fin rays D

n

Gill rackers A D n

0.882 0.667 o-2 o-2

51 24

0.731 0.417 o-2 o-2

52 24

1.192 0.652 o-4 o-2

52 24

Esva sample

0.286 t= 1.46”.“. tz2.90” o-1 7

0.286 tz2.09’ t=2.16* o-1 n

0.286 tz3.14” t = 4.06” O-l 7

Character Horn. Het.-1+2 Horn. vs. Het.-1+2

Pectoral A 0.813 0.364 t=1.74”.” fin rays D o-3 o-1

n 16 11

Pelvic A 0.625 0.455 t=0.85”.” fin rays D o-1 o-1

n 16 11

Gill rackers A 1.182 1.000 t = 0.67”,” D o-2 o-2 n 11 10

Norway sample

Character Horn. Het.1 Het.-2 Horn. vs. Het.-1 Horn. vs. Het.-2

Pectoral A 0.74 fin rays D o-2

n 62

0.68 0.29 t = 0.40”.“. t=2.03’ o-2 o-1

31 I

Pelvic A 0.42 fin rays D o-3

n 62

Gill rackers A 1.39 D o-3 n 62

0.39 0.29 t = 0.24”,“. t = 0.65”.“, o-2 o-1

31 7

1.29 0.48 t = 0.56”.“. tz4.23”

o-3 O-l 31 7

“0.4 G. RLANCO E?’ AL.

Scotland sample

Chararter Horn. Het.-1 Het.-2 + 3 Horn. vs. Het.-1 Horn. vs. Het.-2+X

Pectoral A fin ravs I)

II

Pelvic D

fin rash A ,I

Gill rackers A D 11

0.625

0 3

56

1.375 1.069 0.600 r = 1.76”.” t=3.70” o-4 O-2 O-2

56 ‘9 15

Wi86 0.3

29

0.533 lzO.21” t = 0.47” * I)- 2

15

A = average asymmetry estimated as mean value of [ right side-left side 1. I) = minimum and maximum values of deviation from bilateral symmetry. n = sample size. Horn. = individuals homozygous for the five polymorphic loci. Het.=individuals heterozygous for one (Het.-1). two (Het.-2), 1 or 2 fHet.-1+2), 2 or 3 (Het.- 2 + 3 ) loci, whichever they are. Mean values of asymmetry (A) were compared with a t-test corrected for unequal variances. ‘PC 0.05: “Pi 0.01: “.’ not significant.

( Yella), there was a significant negative correlation between these values. Fur- thermore, the sign of the correlations coefficient was negative in all the anal- yses (sign test, P< 0.05). The finding of non-significant differences in the Esva population could be due to the smaller size of the sample (rz=21). These re- sults support the alternative hypothesis that there is a negative correlation between the proportion of heterozygous loci and the proportion of asymmetric characters.

In addition, we examined in each sample the relationship between the num- ber of heterozygous loci and the global magnitude of the asymmetry per indi- vidual. The global magnitude of asymmetry per individual was computed as the sum of the values of the differences of the three bilateral characters, cor- rected for scaling effect (Soul&, 1967), by the use of normalization of data. Spearman’s rank correlation was used as a comparison test between the num- ber of heterozygous loci and the magnitude of asymmetry. The correlation was negative in all cases and statistically significant in three out of four popula- t,ions under study (Sella: r= -0.33; PcO.01, Nor.: r= -0.21; PcO.05 and Sco.: r= - 0.22; P < 0.05). Correlation was still not significant in the Esva popula- tion (r= -0.18).

In general articles of this type discuss whether this relationship between heterozygosity and less asymmetry is associated with certain individual loci or

DEVELOPMENTAL STABILITY OF HETEROZYGOTES IN SALMO SALAR 205

TABLE 2

Number of heterozygous loci and asymmetric characters in each population

No. of asymmetric characters

No. of heterozygous loci

0 1 2 3

Sella (natural sample) 0 1 2 1 1

1 I 10 3 1 2 30 8 1 3 15 4

r= -0.405; t+,, = -3.99; PcO.01

Esva (natural sample) 0 1 4 3 1 2 2 4

3 5 2

r= -0.239; tcrzO,= - 1.073 n.s.

Nonvay (hatchery sample ) 0 2 2 3 1 17 9 1

2 29 14 3 3 14 6 -

r= -0.204; f,r=,j,= -2.064: PcO.05

Scotland (hatchery sample) 0 2 3 3 1

1 25 13 5 1

2 21 10 4

3 8 3 1

r= -0.223; fcrcO,= -2.264; PiO.05

with a general heterozygous effect. We will now examine the amount of asym- metry for homozygotes and heterozygotes at all loci in which there were over five heterozygotes, in each population (Table 3). In all cases, heterozygotes had a smaller mean of asymmetric traits than homozygotes for that locus, but, the difference between the two groups was significant only for the locus Sdh- 1 in the Sella population. This allows us to discard the hypothesis that the correlation found is due only to heterozygosity of that locus. In any case, and to verify this point, we eliminated Sdh-1 from the data in that sample to de- termine if the overall effect was due only to heterozygosity at this locus. The correlation between proportion of heterozygous loci per fish and the proportion of asymmetric characters per individual remained significant (F= -0.30, t = - 2.71, P-c 0.01)) indicating that in this population heterozygosity in gen-

206 G. BLANC0 ET AL.

TABLE 3

Differences of mean asymmetry between homozygotes and heterozygotes for each locus

Loci Populations

Sella Esva Norway Scotland

Mdh-3

Aat-

Sdh-1

+0.47= + 0 v7”.s. .u + 0.40”.“.

+ 0.41”.” +0.17”.“. +0.21= + 0.40”.“. + 0.94” + 0.44”.“. l tO.21”.“.

“P< 0.01; “.‘, not significant.

era1 and heterozygosity at one specific locus are associated with increased de- velopmental stability. These results are in accordance with previous data cited by other authors. In this sense, Blanc0 and SEinchez (1986), working with syn- thetic populations of D. melunogaster, with varying degrees of heterozygosity, stated that the observed phenomenon is an expression of a general heterozy- gous effect. Within populations, other authors have found in fish (Leary et al., 1983,1984 ) and insects (Blanco, 1988) that heterozygosity in general and het- erozygosity at specific loci are associated with increased developmental stability.

In all cases our results support Lerner’s hypothesis that more heterozygous individuals within a population have increased developmental stability.

Most of the studies on fluctuating asymmetry in relation to allelic isozyme heterozygosity have involved comparisons of populations rather than of indi- viduals within populations. In our case, when the average enzymatic values of each sample are related to the average values of the estimated magnitude of asymmetry for these samples, the results indicate, again, a positive correlation between heterozygosity and asymmetry; as the average heterozygosity of the population increases, the average value of the magnitude of asymmetry de- creases and the correlation is significant (r = - 0.95, t = 4.64, P < 0.05 ) . How- ever, this type of interpopulational study has been criticized because, as pointed out by Zouros and Foltz ( 1987 ), if the objective is to study the direct effect on developmental stability of heterozygosity at the actual enzyme loci, the best approach would be to study heterozygosity and bilateral asymmetry in individ- uals from the same population, because historical and stochastic events, pop- ulation breeding structure, and local selection regimes have a much higher impact on interpopulation comparisons and may accidentally inflate or deflate the correlation between enzyme heterozygosity and fluctuating asymmetry.

Several recent papers have emphasized the importance of maintaining ge- netic variation in cultured stocks of fish ( Allendorf and Phelps, 1980; Ryman and Stahl, 1980; Cross and King, 1983; Kincaid, 1983). Genetic variation is the primary biological resource in the survival and artificial propagation of any species (FAO, 1984). Loss of genetic variation in hatchery stocks of salmonids

DEVEMPMENTAL STABILITY OF HETEROZYGOTES IN SALMO SALAR 207

has harmful effects on a variety of important traits: survival of eggs and young, growth rate, feed conversion efficiency, and body shape (see review in Kincaid, 1983). Furthermore, the loss of genetic variation reduces the ability of a stock to adapt to different environmental conditions. These problems are especially important for hatchery stocks that are used to help found or sustain natural populations (Ryman and Stahl, 1980).

The results of this study confirm that heterozygotes at enzyme loci tend to be less asymmetric than homozygotes, and we found a negative correlation between heterozygosity and asymmetry in all populations, that was significant in three out of the four populations analysed. This relationship indicates that even the relatively small differences in heterozygosity among individuals that can be distinguished with electrophoresis have a detectable effect on develop- mental stability.

Our results, as well as those obtained by other authors, indicate that fluc- tuating asymmetry is sensitive enough to detect differences in developmental stability between individuals with different amounts of heterozygosity at iso- zyme loci. On the other hand, results with gynogenetic diploid rainbow trout confirm that the loss of heterozygosity in inbred fish does produce detectably less developmental homeostasis as measured by fluctuating asymmetry (Leary et al., 1985a,b). Thus, fluctuating asymmetry provides a potentially valuable measure of the loss of heterozygosity in fish populations.

ACKNOWLEDGEMENTS

This work was financed by public funds of the CAICYT (15/&l). We are grateful to the Consejeria de Agricultura y Pesca de1 Principado de Asturias, and to Sociedad Asturiana de Pesca, for their collaboration in obtaining the samples.

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