POPULATIO GEETICS OF THE DOVER SOLE, SOLEA SOLEA

(LIAEUS, 1758) (TELEOSTEI: SOLEIDAE)

Thesis submitted in accordance with the requirements of the University of Liverpool

for the degree of Doctor of Philosophy

by

Athanasios Exadactylos B.Sc. (Hons), M.Phil.

Port Erin Marine Laboratory

School of Biological Sciences
University of Liverpool
Port Erin
Isle of Man September 1997
To my parents with all my heart

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 COTETS

ACKOWLEDGEMETS ........................................................................................... vi

ABSTRACT .................................................................................................................... vii

CHAPTER OE: GEERAL ITRODUCTIO ..................................................1

1.1 Molecular systematics .............................................................................................2
1.2 Genetics in fisheries management and conservation ..............................................4
1.2.1 Historical perspective .....................................................................................4
1.2.2 Fishery management ......................................................................................7
1.2.3 Population genetics in fishery management ..................................................8
1.2.4 Conservation genetics .................................................................................10
1.3 Genetic management of culture stocks .................................................................11
1.3.1 Quantitative genetics in aquaculture propagation ........................................13
1.3.2 Association of heterozygosity with fitness-related traits ............................15
1.4 Future studies using genetic markers ....................................................................17
1.5 Aims of the study ..................................................................................................21
CHAPTER TWO: GEERAL MATERIALS AD METHODS .........................23
2.1 Introduction ..........................................................................................................24
2.2 Sample collection .................................................................................................24
2.3 Electrophoresis ......................................................................................................26
2.3.1 Preparation of the starch gels .......................................................................26
2.3.2 Preparation of the tissue samples ................................................................27
2.3.3 Buffer systems and electrophoretic set-up ..................................................27
2.3.4 Staining and agar overlay techniques ..........................................................28
2.3.5 Scoring and genetic interpretation of zymograms ........................................32
2.3.6 Statistical analysis ........................................................................................32
CHAPTER THREE: ALLOZYME VARIATIO AD GEETIC ITER-
RELATIOSHIPS BETWEE SPECIES OF THE FAMILIES
PLEUROECTIDAE, SCOPHTHALMIDAE AD SOLEIDAE .............................36
3.1 Introduction ...........................................................................................................37
3.2 Materials and Methods ..........................................................................................42
3.2.1 Sample collection sites .................................................................................42
3.2.2 Analysis of electrophoretic variation ...........................................................45
3.3 Results ...................................................................................................................47
3.3.1 Allelic frequencies .......................................................................................47
3.3.2 Panmixia ......................................................................................................51
3.3.3 Genetic heterozygosity .................................................................................53
3.3.4 Genetic distance ...........................................................................................53
3.4 Discussion .............................................................................................................60
3.4.1 Genetic relationship between species ..........................................................62

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CHAPTER FOUR: POPULATIO STRUCTURE OF THE DOVER SOLE,
SOLEA SOLEA L., I THE ATLATIC AD MEDITERRAEA ......................67
4.1 Introduction ...........................................................................................................68
4.2 Materials and Methods ..........................................................................................70
4.2.1 Sampling protocol ........................................................................................70
4.2.2 Statistical analysis ........................................................................................73
4.3 Results ...................................................................................................................75
4.3.1 Allele frequencies ........................................................................................75
4.3.2 Conformity to Hardy-Weinberg expectations .............................................83
4.3.3 Heterozygosity .............................................................................................83
4.3.4 Population structure .....................................................................................91
4.3.5 Genetic distance ..........................................................................................94
4.4 Discussion .............................................................................................................98
4.4.1 Genetic variability within populations .........................................................98
4.4.2 Differentiation between populations ..........................................................100
4.4.3 Differentiation within basins ......................................................................103
CHAPTER FIVE: PCR (RAPD) AMPLIFICATIO OF TOTAL GEOMIC
DA OF THE DOVER SOLE, SOLEA SOLEA L., FROM THE ATLATIC .... 105
5.1 Introduction .........................................................................................................106
5.2 Materials and Methods ........................................................................................108
5.2.1 Sample tissue collection and digestion ......................................................108
5.2.2 DNA extraction and quality control ...........................................................109
5.2.3 RAPD-PCR procedure ...............................................................................111
5.2.4 Data analysis ..............................................................................................112
5.3 Results .................................................................................................................115
5.4 Discussion .......................................................................................................... 129
5.4.1 Comparison of RAPD and allozyme data ..................................................129
5.4.2 DNA polymorphism between populations .................................................130

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CHAPTER SIX: GROWTH RATE AD HETEROZYGOSITY I EARLY
LIFE HISTORY OF THE DOVER SOLE, SOLEA SOLEA L. ............................... 134
6.1 Introduction .........................................................................................................135
6.2 Materials and Methods ........................................................................................137
6.2.1 Incubation conditions .................................................................................138
6.2.2 Rearing conditions .....................................................................................139
6.2.3 Feeding .......................................................................................................141
6.2.3.1 Hatching procedure of Artemia nauplii cysts .................................141
6.2.3.2 Live food enrichment ....................................................................142
6.2.3.3 Continuous culture of the algae Isocrysis galbana var tahitian .....143
6.2.4 Sampling procedures ..................................................................................143
6.2.5 Statistical methods .....................................................................................145
6.2.6 Analysis of electrophoretic data .................................................................146
6.3 Results .................................................................................................................148
6.3.1 Age and growth ..........................................................................................148
6.2.2 Genetic diversity ........................................................................................165
6.2.3 Heterozygosity and gowth .........................................................................169
6.4 Discussion ...........................................................................................................174
6.4.1 The relationship between growth rate and multiple and single locus
heterozygosity .....................................................................................................176
CHAPTER SEVE: COCLUSIOS ......................................................................180
7.1 Systematics ..........................................................................................................181
7.2 Population genetics .............................................................................................182
7.3 Quantitative genetics ............................................................................................185
REFERECES ..............................................................................................................188

APPEDICES ...............................................................................................................221
Appendix I .................................................................................................................... i
Appendix II ............................................................................................................... viii
Appendix III ............................................................................................................... xvi
Appendix IV............................................................................................................. xviii
Appendix V ............................................................................................................... xix
Appendix VI ............................................................................................................... xx
Appendix VII ............................................................................................................ xxii
Appendix VIII ........................................................................................................... xiv
Appendix IX ........................................................................................................... xxvii
Appendix X .............................................................................................................. xxxi

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 ACKOWLEDGEMETS

I would like to thank my supervisors, Reader J. P. Thorpe and Dr. A. J. Geffen

for their encouragement, advice, support and guidance throughout the period of this
study. Other academic, administrative and technical staff at the Port Erin Marine
laboratory have also assisted in various ways to the success of this work. Special
thanks to Audrey for the late support in ‘dosh’ for the RAPDs, but also to John for the
‘outrageous’ talks in the early morning hours.
I feel in debt to F. Lagardere and the crew at La Rochelle; A.D. Rijnsdorp and
colleagues at RIVO-DLO; the fisheries group at CEFAS, Lowestoft; P. Newton and
DANI at Belfast; our crew from R.V. Roagan, B.R. Howell at CEFAS, Conwy; D.S.
Danielssen at Fl∅devigen, as well as the local fishermen in Thessaloniki, for their help
in sample collection (still having nightmares about it!).
My thoughts and thanks also go to all those people that helped make my stay in
this remote island enjoyable enough. It is difficult to start, or finish; B.F. society or
not; Wanni and his little Sanuri (me, Mr. Blooby!), Eduardo-the man, Amina-the girl,
Terry and Salma and Sammy and Sara and..., Katerina, Puri, Bairbre, Fiona, Harry,
Kate, Seanny, Kevin (tea-man), Laura, Moises, Federica, Stuart, Zhang, Diana, Phil,
Smoo, Kieran, Candace, David, Mr. Chul, Mark (a Volvo?), Andy (Scot), Alex and
Sarah, Doug and his ‘draculini’, Javier, Felipe, Alexis, the lads from my ‘hoop’ days,
and my ‘guys’ back home for being there. Above all, I have had the good fortune of
making true friends who have been extremely encouraging during the depression
‘ages’; Erema (for these comprehensive plans in the black hole) and Henar (missed
her a lot at the final stages of the thesis).
Many, many thanks also to Meni, Panagiota and T. Alifakiotis, for opening their
doors to me. Especially to Panagiota for counting on me even during those bad
patches.
The helpful comments made by Dr. A.D. Rogers and Dr R.D.M. Nash, external
and internal examiner respectively, are gratefully acknowledged.
This study was funded by the Greek Scholarship Foundation and I am grateful
to the administrators at the Committee in Athens for their assistance.
Do not forget my beloved sister!!!

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 Population Genetics of the Dover sole, Solea solea (Linnaeus, 1758)
(Teleostei: Soleidae)
Athanasios Exadactylos
ABSTRACT
Dover sole, Solea solea L. was selected for this study because of its commercial importance and
potential for aquaculture. Molecular genetic techniques have been applied to evaluate stock differences
in S. solea and the implications of the results in fisheries management, fisheries conservation and
aquaculture propagation are discussed.
Allozyme electrophoresis was used as a molecular tool to establish the evolutionary inter-
relationships between 17 populations (Atlantic and Mediterranean) of the most common seven non-
sibling species within the families Pleuronectidae, Scophthalmidae and Soleidae. It seems that
flatfishes as a group show higher levels of genetic variation (mean polymorphism, observed
heterozygosity) than other fish species. For most of the 17 populations studied, values of genetic
identity for all the pairwise comparisons fell within the range of values for populations within species,
between both congeneric species and between genera, described in the literature. The data indicate the
regrouping of the plaice and the flounder into a single genus Pleuronectes and the retention of the dab
in a separate genus Limanda. The high level of genetic divergence between Dover sole and thickback
sole could be explained by the latter’s life cycle, which differs from the other flatfishes in having an
entirely offshore development. Results also indicate the occurrence of Pleuronectes flesus luscus as a
subspecies of Pleuronectes flesus in the eastern Mediterranean. The families Pleuronectidae and
Scophthalmidae were found to be sister taxa; approximately 5 M. yrs ago the families Pleuronectidae
and later Soleidae evolved from the ancestral Scophthalmidae.
The present work was also an attempt to address a spatial-scale study of the genetic structure of
the Dover sole, using allozymes and randomly amplified polymorphic DNA (RAPD) as genetic
markers. The essential genetic structure conclusions from both markers were in general agreement,
although RAPD markers detected more variation (% polymorphism, heterozygosity) in the sole
populations sampled. The observed heterozygote deficiencies (high FIS values) may have resulted from
the chance occurrence of single very rare homozygotes for rare alleles. Nevertheless, no significant
deviation from the Hardy-Weinberg equilibrium was observed, hence, there was little reason to reject
an hypothesis of random mating. There was significant heterogeneity in allele frequencies, hierarchical
FST and non-hierarchical FDT estimates among populations and between groupings (north-west to
south-east), indicating the level of genetic diversity of the species. The progressive decline in allozyme
allelic heterozygosity could be interpreted as a possible consequence of a population bottleneck,
founder effects, genetic drift, or a combination of these. Evidence in favour of the genetic divergence
between populations and regions was shown by their geographic clustering in the relevant dendograms.
A strong similarity (high pairwise gene flow values) between the Bay of Biscay and the German Bight
populations, and the three Irish Sea populations with the East Anglian population, indicated a probable
movement of migrants, or a strong capability of passive exchanges during pelagic stages through the
English Channel. Nevertheless, there was no evidence of significant correlation of genetic with
geographic distance. This apparent absence of isolation-by-distance suggests that S. solea may not be
at genetic equilibrium.
Finally, collecting larvae at intervals during their developmental period (laboratory-based rearing
experiments) generated information about patterns of growth in two sole batches derived from different
broodstocks. The significant variability in larval standard lengths between the different experimental
treatments (stocking densities) suggested that larvae initially at one or both extremes of length may have
been eliminated from the populations, resulting in greater uniformity in size and weight among older
larvae. This mortality did not appear to be genotype-dependent, since allele frequencies did not deviate
from those expected under the Hardy-Weinberg equilibrium. Genetic diversity estimates (%
polymorphism, heterozygosity, genetic distance, heterogeneity of allele frequencies, FST analyses) were
either considerably lower than those of wild sole populations, or not significantly different from them,
demonstrating a loss of genetic variation and a high degree of genetic similarity between the two
hatchery stocks, respectively. Furthermore, there was no evidence in this study that the degree of multi-
locus or single-locus heterozygosity correlates well with growth rate (one of a number of physiological
attributes) in S. solea, which agrees with the observation that such correlation is not expected in
populations where there is no heterozygote deficiency.
These results indicate the role of ecological, biological and evolutionary structuring mechanisms
in determining the population structure of S. solea.

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