Developmental stability and genetic heterozygosity
in wild and cultured stocks of gilthead
sea bream (
Sparus aurata)
J. Palma*
½, J.A . A larcon
O, C. A lvarez
O, E. Zouros
P, A. Magoulas
Pand J.P. Andrade*
*CCMar, FCMA, Universidade do Algarve, 8000^810 Faro, Portugal.ODepartment of Cell Biology and Genetics, Faculty of Sciences, University of Malaga, Campus Universitario Teatinos, 29071 Malaga, Spain.
PInstitute of Marine Biology of Crete, PO Box 2214, Gr 71003 Iraklion, Greece.
Department of Biology, University of Crete, PO Box 1470, Gr 71110, Iraklion, Greece.
½Corresponding author, e-mail: [email protected]
The present study checks on the validity of the hypothesis that heterozygosity and the £uctuating asymmetry (FA), common measure of the developmental stability, are linked in populations of wild and cultured stocks ofSparus auratafrom ¢ve countries. Muscle and liver samples were analysed for variation in 26 allozymes and three microsatellite loci. Pectoral ¢n rays and upper and lower gill rakers of the ¢rst branchial arch were counted on the left and on the right sides of each ¢sh. Fluctuating asymmetry existed in the majority of the samples although their values were consistently low, (0.3050.147), but higher in the cultured samples. The allozyme heterozygosity values were always high, but lower in the cultured samples. The microsatellite DNAanalysis produced similar results. Heterozygosity was higher in cultured individuals (except for the Greek samples). These ¢ndings seem to be early evidence that the reared samples are losing some genetic variation, especially due to the loss of the rarest alleles (which were present in the wild populations). Genetic drift, probably caused by propagation practices, is most likely responsible for the decrease of the genetic variation. No distinct pattern of geographic separation was identi¢ed.
INTRODUCTION
The morphology of an individual is produced by the developmental process that transforms the genotype into the phenotype (VÖllestad & Hindar, 1997). The ability of development to produce a determined phenotype despite eventual disturbing factors is de¢ned as developmental stability (Leary et al., 1992). Fluctuating asymmetry, small random departures from bilateral symmetry (Markow, 1995) has been used as a measure of developmental stability of bilateral, normally symmetrical morphological traits (Palmer & Strobeck, 1986; Zakharov, 1992). Fluctu-ating asymmetry quanti¢es the di¡erences between the two sides in various morphological traits: the larger the FA, the lower the developmental stability. The concept is that individuals of low ¢tness cannot control their development precisely, and consequently more often develop di¡erent phenotypes on both sides (Windig & Nylin, 2000). Both intrinsic (genetic) and extrinsic (environmental) factors may in£uence developmental stability (Wilkins et al., 1995). Their relative impact can be evaluated by means of FA, which can be easily used by aquaculturists, simply by counting meristic traits.
Fluctuating asymmetry is a suitable indicator of stock condition and heterozygosity levels (Crozier, 1997) in salmonids and other ¢sh (Leary et al., 1984, 1985; Blanco et al., 1990). In recent years, a number of studies have examined the relationship between enzyme hetero-zygosity and FA(e.g. Leary et al., 1983; Crozier, 1997). The results of these studies have supported the hypothesis
that individuals or populations with higher heterozygosity also display higher developmental homeostasis, which is re£ected by lower degrees of bilateral asymmetry of meristic traits.
The main objective of the present work was to evaluate the relationship between FAand multilocus hetero-zygosity, derived from allozyme and microsatellite markers, between wild and cultured gilthead sea bream,
Sparus aurata (L.), collected in ¢ve European countries. The geographic variation of data was also studied.
MATERIALS AND METHODS
Sample collection
Atotal of 229 wild specimens of gilthead sea bream were captured along the coasts of Portugal (N50, Ria de Aveiro Lagoon, PtW) (W, wild), Atlantic Spain (N48, Cadiz, SpAtW), Mediterranean Spain (N51, Alicante, SpMW), Italy (N40, Trieste, ItW) and Greece (N40, Mesologgi Lagoon, GrW) (Figure 1). Addition-ally, 273 individuals were obtained from aquaculture farms located in France (N23, Oleron Island, FrR) (R, reared), Portugal (N50, Tavira, PtR), Atlantic Spain (N50, Cadiz, SpAtR), Mediterranean Spain (N50, Murcia, SpMR), Italy (N50, Trieste, ItR) and Greece (N50, Leros Island, GrR) (Figure 1). Original sample sizes ranged between 40 and 51 ind. However, the French reared sample was reduced to 23 ind, because in 17 speci-mens the number of pectoral ¢n rays could not be scored
on one of the sides. All individuals showed adult morphology and were rather similar in size. Both wild and reared seabreams were collected between 1996 and 1997.
After collection, animals were transported to the laboratory frozen on ice for dissection and further meristic analysis. Apiece of liver, dorsal muscle and the intact eye from each individual were stored frozen in labelled tubes for allozyme screening. Apiece of muscle was stored in 70% ethanol for microsatellite DNA analysis.
Allozyme electrophoresis
For allozyme electrophoresis, frozen tissues were subjected to no more than three freezing and thawing cycles to obtain a cell lysate, which was run through a horizontal starch gel. Electrophoretic protocols, staining procedures and genetic interpretation of zymogram patterns and locus designation were done according to Reina et al. (1994). Sixteen enzymatic systems were used in the sample analysis (Table 1). The individual and sample heterozygosity (heterozygous loci in each animal/ number of loci of each animal) was calculated for all samples.
Microsatellite DNA analysis
To estimate heterozygosity values three microsatellite loci were screened: SA26, SA32 and SA41b, (SA, Sparus aurata) with EMBL library accession numbers Y17266, Y17264 and Y17262, respectively.
DNAextraction from each individual was performed according either to the standard proteinase-K protocol (Sambrook et al., 1989) or the salt-extraction technique of Miller et al. (1988). 10^100 ng of these DNAs were used in polymerase chain reaction (PCR) reactions (vol. 10mm), containing 0.2 mM of each dNTP, 1mM MgCl2,
0.5mM of each primer, and 0.5 units of Taq polymerase. Asmall fraction of the reverse primer was end-labelled prior to ampli¢cation. Each ampli¢cation included seven cycles with denaturation at 948C, annealing at 528C and extension at 728C, and 28 cycles under the same condi-tions, except that the denaturation temperature was 888C. All cycles lasted for 30 s. An aliquot of the PCR products (5ml) was run in 6% polyacrylamide denaturing sequencing gels (2.5 h at 60W). DNAbands were visua-lized by autoradiography. The process was performed according to Batargias et al. (1999).
Fluctuating asymmetry
Fluctuating asymmetry was assessed by counting three bilateral meristic characters: pectoral ¢n rays (PFR), and gill rakers on the upper and lower ¢rst branchial arches. Gill rakers on the ¢rst branchial arch were counted on the arch's upper (epibranchial) and lower (ceratobranchial) limb. These characters were chosen because they could be easily and accurately counted. The raker in the angle between the upper and lower limb was counted in the lower limb. Gill rakers were divided into upper and lower sectors, because they have been shown to vary indepen-dently (Leary et al., 1983), and hereafter will be referred to as UGR and as LGR. Other bilateral characteristics
Figure 1. Sampling locations: Greece (1a, Mesologgi Lagoon (wild sample), 1b, Leros Island (reared sample)); Italy (2, Trieste, Italy (both wild and reared samples)); Mediterranean Spain (3, Murcia (both wild and reared samples)); Atlantic Spain (4, Cadiz (both wild and reared samples)); Portugal (5a, Tavira (reared sample), 5b, Ria de Aveiro lagoon (wild sample)), France (6, Oleron Island, reared sample).
such as the pelvic ¢ns were not scored because they do not show variation in the Sparidae ¢sh.
All individuals of the cultured sample from Greece had an abnormality and did not present gill rakers in the ¢rst branchial arches. Thus, these were only scored for the pectoral ¢n rays.
Statistical analysis
Four steps were followed in the analysis of £uctuating asymmetry (FA) data: (i) a two-way analysis of variance (ANOVA) tested the data, regardless of the right^left orientation. Larger values were placed in a ¢rst column and the smaller ones in a second. No statistical di¡erence between columns indicates that asymmetry is not di¡erent from bilateral symmetry. Astatistical di¡erence indicates that asymmetry exists. (ii) Atwo-way ANOVA tested the data in its original orientation with respect to right and left sides (Palmer & Strobeck, 1986). No statis-tical di¡erence indicates non-directionality. Columns were tested for normality and variance of homogeneity (Sokal & Rohlf, 1981). (iii) The distribution of the signed asymmetries was tested for normality (Palmer & Strobeck, 1986), normality indicate no antisymmetry; and (iv) if the assumptions of the previous points were veri¢ed the FA was calculated and expressed as variance, var(Ai), were Ai(Ri7Li), (Ai, asymmetry of a particular character for individual i; Ri, counts on the right side; Li, counts on left side). The above procedures were performed as suggested by Pomory (1997) for determining the pattern and type of asymmetry. The FAindices were correlated with the allozyme and microsatellite heterozygosity levels.
The Shapiro^Wilks test (Conover, 1980; Zar, 1984) was used to access normality of data.
Within-sample relationships between heterozygosity (allozyme and microsatellite loci) and asymmetry (FA) were examined at the individual level using the Spearman rank correlation test (Conover, 1980). Signi¢-cance of correlation coe¤cients was corrected using the Bonferroni method (Snedecor & Cochran, 1982). Anull
hypothesis, stating that heterozygous individuals have the same level of bilateral symmetry as homozygous indivi-duals was tested in this analysis.
Fluctuating asymmetry and heterozygosity were compared among samples using the Wilcoxon signed rank test (Conover, 1980; Zar, 1984). Homogeneity among samples was tested using w2-test (Conover, 1980; Zar,
1984). Statistical analysis was performed using the soft-ware package SPSS1for Windows.
RESULTS
Allozyme heterozygosity
From the 24 loci screened, 11 proved polymorphic (EST, GPI-2, IDDH, IDHP, LDH-2, MDH-2, MDH-3, PGM, PGDH, SOD-1, SOD-2). Among these, ¢ve were polymorphic in all the samples (EST, GPI-2, IDHP, PGM, PGDH).
The percentage of heterozygous ¢sh observed for each allozyme, as well as the observed heterozygosity (Ho), are presented in Table 2. Individually, ¢sh were heterozy-gous to a maximum of six loci.
Microsatellites
High percentages of heterozygous individuals were found for the majority of the samples (Table 3). Nine samples presented 100% of heterozygous ¢sh for at least one MS locus.
The average observed heterozygosity values varied between 0.78 (PtR) and 0.91 (SpMR) in the reared samples, and between 0.61 (ItW) and 0.92 (GrW) in the wild samples (Table 3).
Asymmetries and £uctuating asymmetry Asymmetries
Table 4 shows the percentage of ¢sh with asymmetries and the percentage of ¢sh with at least one asymmetry (A1). The Italian sample (R) was the most asymmetric (92%), while the Greek sample (W) was the least
Table 1. List of enzyme systems.
E.C. no. Tissue Bu¡ers No. of loci
Adenilato kinase (AK-1 and AK-2) 4.2.1.3. Muscle CTC 2
Adenosine desaminase (ADA) 3.5.4.4. Liver RID 1
Alcohol dehydrogenase (ADH) 1.1.1.1. Liver CTC 1
Diaforase (DIA-1) 1.6.2.2. Liver RID 2
Esterase (EST) 3.1.1.*. Liver RID 1
Glucose-6-phosphate dehydrogenase (GPI1 and GPI2) 5.3.1.9. Muscle RID 2 Glicerol-3-phosphate dehydrogenase (G3PDH) 1.1.1.8. Muscle CTC 1
Iditol dehydrogenase (IDDH) 1.1.1.14. Liver CTC 1
Isocitrate dehydrogenase (IDHP) 1.1.1.42. Liver CTC 1
Lactate dehydrogenase (LDH-1, LDH-2 and LDH-3) 1.1.1.27. Eye RID 3 Malate dehydrogenase (MDH-1, MDH-2 and MDH-3) 1.1.1.37. Muscle CTC 3
Malic enzyme (MEP-1 and MEP-2) 1.1.1.40. Muscle CTC 2
Phosphoglucomutase (PGM) 5.4.2.2. Muscle RID 1
6-phosphogluconate dehydrogenase (PGDH) 1.1.1.44. Liver CTC 1 Superoxidase dismutase (SOD-1 and SOD-2) 1.15.1.1. Liver RID 2
Xantine dehydrogenase (XDH) 1.2.1.37. Liver CTC 1
asymmetric (37.5%). The Spanish reared samples (SpAtR, SpMR) and the Italian wild sample (ItW) also presented high levels of asymmetry, respectively 68%, 64% and 65%.
Fluctuating asymmetry
Pectoral ¢n rays (PFR) and the lower gill rakers (LGR) in the GrW failed the statistical procedure (¢rst step), and were excluded from further analysis. The LGR of the SpAtR and PtW also failed, not presenting FA (second step), and were also excluded (Table 4).
Fluctuating asymmetry varied between 0.096 (SpAtW) and 0.591 (SpMR) for PFR; 0.189 (PtW) and 0.551 (SpAtR) for UGR, and 0.134 (FrW) and 0.5 (ItR) for LGR (Table 4). Results of the Shapiro^Wilks test indi-cated that meristic traits were normally distributed.
No signi¢cant correlations (Spearman rank correlation test; P50.005) were found between the heterozygosity (both allozymes and microsatellites).
Wild samples were quite similar, with a marginal degree of heterogeneity. In contrast, there was a consider-able di¡erence in the degree of asymmetry among the
Table 2. Percentages of heterozygous ¢sh observed for each allozyme screened for all samples. Monomorphic loci are not presented.
FrW PtR PtW SpAtR SpAtW SpMR SpMW ItR ItW GrR GrW
EST 60.9 46 56 36 62.5 38 49 46 45 32 52.5
GPI2 52.2 34 100 52 35.4 82 41.2 20 32.5 34 42.5
IDDH 0 0 0 0 0 0 3.9 0 2.5 0 0
IDHP 39.1 48 38 32 31.3 68 45.1 62 42.5 38 42.5
LDH-2 0 0 0 0 0 0 2 0 0 0 0
MDH-2 0 0 0 0 0 0 5.9 8 0 0 0
MDH-3 0 0 0 0 2.1 0 2 0 0 0 0
PGM 56.5 36 56 68 52.1 82 74.5 44 55 38 65
PGDH 43.5 48 36 20 58.3 62 41.2 48 55 56 30
SOD1 0 0 0 0 0 0 5.9 0 0 0 0
SOD2 4.3 20 8 10 6.3 0 2 2 0 18 0
Ho 0.0935 0.0912 0.0919 0.0846 0.1011 0.1392 0.0913 0.0878 0.091 0.0823 0.0882
Ho, observed heterozygosity.
Table 3. NHF is the percentage of heterozygotic ¢sh observed for each one of the three MS loci. MS1 is the percentage of ¢sh in each sample that was heterozygotic for at least one MS loci.
FrW PtR PtW SpAtR SpAtW SpMR SpMW ItR ItW GrR GrW
Sa26 75 82.4 92.1 92.1 77.7 89.7 77.4 95.6 63 86 87.5
NHF Sa32 93.8 94.1 39.3 76.3 77.7 96.6 67.7 86.7 51.9 97.2 87.5 Sa41b 87.5 82.4 60.7 81.6 88.9 79.3 87.1 77.8 92.6 88.9 96.7
MS1 100 100 93 100 100 100 97.2 100 100 100 100
Sa26 0.83 0.64 0.76 0.92 0.8 0.93 0.78 0.96 0.57 0.8 0.89
Ho Sa32 0.97 0.98 0.55 0.85 0.78 0.96 0.63 0.85 0.41 0.98 0.89
Sa41b 0.74 0.73 0.63 0.78 0.91 0.85 0.82 0.78 0.86 0.85 0.97 pooled MS 0.85 0.78 0.65 0.85 0.83 0.91 0.74 0.86 0.61 0.88 0.92
Ho, heterozygosity observed.
Table 4. Percentage of ¢sh that were asymmetric for each one of the studied characteristics in each sample. A1 is the percentage of asymmetric ¢sh for at least one characteristic.
FrW PtR PtW SpAtR SpAtW SpMR SpMW ItR ItW GrR GrW
P.F.R. 21.8 20 18 26 25 22 9.8 60 20 48 7.5
U.G.R. 13 20 16 24 31.3 30 21.6 50 32.5 * 7.5
L.G.R. 39.1 20 20 36 33.3 40 25.5 42 40 * 22.5
A1 56.5 50 42 68 64 58.3 47.1 92 65 48** 37.5
P.F.R. 0.209 0.202 0.183 0.423 0.096 0.591 0.255 0.1318 0.4 0.499 0.154**** FAU.G.R. 0.391 0.202 0.189 0.551 0.256 0.529 0.404 0.473 0.408 * 0.215 L.G.R. 0.134 0.204 0.149*** 0.219*** 0.34 0.366 0.383 0.5 0.317 * 0.076****
*, these characteristics were not scored in the GrR sample; **, based in just one characteristic; ***, failed the statistical procedure (¢rst step); ****, failed the statistical procedure (second step).
reared samples. w2-tests for analysis of homogeneity
among samples were barely signi¢cant (Table 5).
When the allozyme heterozygosity was compared in the reared individuals, the SpMR was signi¢cantly di¡erent from all the other samples (Wilcoxon signed rank test; P50.005). No signi¢cant di¡erences were found among the wild populations (Wilcoxon signed rank test; P50.005). Similar results were obtained for the microsatellite heterozygosity in the reared animals. In the wild animals, the Greek sample was signi¢cantly di¡erent from the Portuguese and the Italian samples (Wilcoxon signed rank test;P50.005).
DISCUSSION
In this study, allozyme heterozygosity showed high values, in contrast, the FAlevels were quite low which is in agreement with the results reported by Palmer & Strobeck (1986) and Crozier (1997) for Atlantic salmon. Nevertheless, the comparative analysis between reared and wild populations suggests that developmental stability appears to be weaker in the reared samples. All reared samples (except for SpMR), presented lower allozyme heterozygosity values and higher values of FA (except for pooled FAin ItW) than the wild samples. This result might indicate a loss of genetic variability of the reared populations. Although microsatellite values were always higher for the reared samples (except for the Greek samples) this could be attributed to the mixed origin of founding populations and/or the renewal prac-tice of the stocks. The allozyme loci usually have only a few alternative states (alleles), they are involved in the functioning of the biochemical pathways of the cell and they are candidate targets of natural selection. Large di¡erences at allozyme loci are not usual among conspe-ci¢cs samples and when seen, they imply very strong selective di¡erences exacted on the population by virtue of di¡erence in their environments, or (more likely) that the populations have been in reproductive isolation from each other for a very long period of time. In contrast, microsatellites are small stretches of DNAmade by tandem repeats of a pair of nucleotide bases and vary from each other because the number of pairs varies widely among homologous sites. As a result, the two
copies an individual receives from each of its parents usually vary, with the consequence that the majority of individuals are heterozygous for each microsatellite (Zouros et al., 1998). Thus, there is a big di¡erence between allozymes and microsatellites in the amount of variation among individuals in population.
These di¡erences between natural and cultivated samples are justi¢ed by the dominance of the rare alleles in the wild populations, which have disappeared in the reared ones through genetic drift e¡ect (Zouros et al., 1998). This fact is related to breeding techniques used in aquaculture, which rely on the same parental brood stock for several generations. Samples showed a predictable result of a high degree of homogeneity among samples.
The majority of the meristic traits did not show high values of FA, which indicates that the mechanisms of developmental stability were present. No destabilization on the development seems to be occurring, except for the Spanish reared samples (both Atlantic and Mediterranean) and the Italian samples (both wild and reared). The higher asymmetry obtained in these samples, could be related to environmental stress. This hypothesis is also related to the fact that the hetero-zygosity values were very similar for all the samples. The higher levels of asymmetry of the Italian samples can be a¡ected by particular abiotic conditions in the northern Adriatic (e.g. temperature, salinity and fresh-water in£ow) (Tomczak & Godfrey, 1994). The values presented by the ItR (highest A1 of all the samples) seem to be enhanced under arti¢cial conditions (due to breeding techniques) what seems to be the case in the wild populations. The high and similar FA obtained for both Spanish cultivated samples might result from exchanges between hatcheries, probably re£ecting common origins. Moreover, animals might be experiencing di¡erent rearing conditions when compared to other countries. On the contrary, the wild populations from France, Portugal, Spain (Atlantic and Mediterranean) and Greece as well as the cultivated populations from Portugal and Greece might re£ect more stable environmental and culturing conditions.
The present study does not clearly indicate a distinct pattern of geographic di¡erentiation for both wild and cultivated samples. In fact, non signi¢cant di¡erences were found for the comparison of the same data. The results obtained for the cultivated samples suggest two main hypotheses: ¢rstly, the exchange of eggs and breeders between aquaculture facilities within countries (as mentioned above) and also between neighbouring countries which is a common practice; and secondly, the production ofSparus auratais still a quite recent technique, and until now, it is not responsible for considerable changes in the genetic pool of cultivated populations.
In the case of the wild samples, the migration of individuals and consequent crossing between neigh-bouring populations is the main reason for their shared characteristics. Considering the dispersal abilities and the high fecundity of the gilthead sea bream, only slight genetic di¡erentiation could a priori be expected.
This work was funded by the EC project AIR3-CT94-1926 and a PhD grant to Jorge Palma (PRAXIS XXI/9441/96). The
Table 5. Results obtained forw2-test.
w2-test
w2 df P
PtR 25.469 13 0.044
SpMR 12.722 12 0.389
Reared SpAtR 25.469 15 0.044
ItR 11.186 15 0.739
GrR 7.789 6 0.254
FrW 5.655 6 0.463
PtW 11.426 8 0.179
Wild SpMW 11.426 8 0.179
SpAtW 15.132 8 0.057
ItW 11.186 15 0.739
authors also wish to thank to Dr Pierick Ha¡ray who provided the French sample. Thanks are due to Dr R. Castilho for her help with data analysis and references. The manuscript bene¢ted from the comments of two anonymous referees.
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