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The use of molecular genetic markers for the study of stock structure of anchovy (Engraulis encrasicolus) in the Mediterranean and adjacent seas

Objective

This project aims to substantiate the population structure of anchovy in the Mediterranean and the adjacent seas, by combining information derived from genetic markers coming from the non-coding portion of nuclear genome and the mitochondrial genome. The project builds on earlier work on RFLP analysis of mitochondrial DNA (mtDNA) variation of anchovy, which revealed a population structure uncommonly complex for a marine species. In order to provide direct evidence about the causes of this pattern of population differentiation, in the context of the present project, more samples of adult individuals from critical areas will be examined, and the mtDNA analysis will be extended to premature stages (larvae). The RFLP haplotypes revealed in the previous study will be also analysed by sequencing the control region of mtDNA. Finally, the analysis will be extended in the nuclear genome by developing and applying highly polymorphic microsatellite markers. Results so far confirmed the relative genetic homogeneity of the Black Sea, especially if compared with Mediterranean, and sequence analysis confirmed the high degree of evolutionary divergence between haplotypes co-existing in the Mediterranean and the Atlantic. An assay for the quick characterisation of the individuals regarding their mtDNA haplotype have been developed, which could facilitate future work for routine stock monitoring.
During the first year of the project seven samples of adult animals were caught from areas which are of critical importance for understanding the population structure and dynamics of anchovy in the Mediterranean and adjacent seas: Black Sea (2), northern and southern Adriatic, south coast of Portugal, and Atlantic coast of Morocco. Three larval samples were collected from southern (Saronikos Gulf) and northern Aegean (near Samothraki island and Strymonikos Gulf).
RFLP analysis of the mitochondrial DNA of the adult samples has been started already and have provided strong evidence that the Black sea is genetically homogeneous for phylad A, both in space and time. Sequencing of the control region of mtDNA has been fully standardised and first and estimates of the sequence divergence between the two phylads and among individuals belonging to the most frequent RFLP haplotype have been obtained.
Two markers (loci) have been developed for microsatellite DNA analysis, and the conditions for scoring have been standardised. In a preliminary test in a sample of 50 individuals these loci were successfully tested and proved to be highly polymorphic: each of them revealed more than 35 alleles.
A quick test for the characterisation of the individuals regarding their phylad has been established. It is based on PCR amplification of the control region of mtDNA and restriction with the endonuclease HincII, which cuts the PCR product of individuals belonging to phylad B, but not that of individuals belonging to phylad A.

DISCUSSION

Some remarkable findings have already been obtained at this early stage of work. Evidence has been provided that the population of Black Sea is rather homogeneous for phylad A, both in space and time. Sequencing of the control region confirmed the high degree of evolutionary divergence between the two phylads, which was evidenced by RFLP analysis. Highly variable microsatellite markers have been developed allowing the extension of the study to nuclear genome. Finally, an easy to perform and quick test for phylad characterisation has been developed. The fact that this test is PCR based and thus can be performed also and the larval stage makes it a valuable tool for monitoring purposes, especially in areas where larval drift may play an important role in recruitment patterns.

REFERENCES

MAGOULAS A., N.TSIMENIDES and E. ZOUROS. 1996. Mitochondrial DNA phylogeny and the reconstruction of the population history of a species: the case of European anchovy (Engraulis encrasicolus). Molecular Biology and Evolution 13(1): 178-190.
INTRODUCTION

Earlier work on RFLP analysis of mitochondrial DNA variation of anchovy (Magoulas et al., 1996), demonstrated that the population in the Black Sea is rather different from that in the neighbouring Aegean, the rest of Mediterranean and the Atlantic. The genetic pattern produced from that study is characterised mainly by the existence of two distinct groups of haplotypes (phylads A and B), the frequency of which varies to a large extent from basin to basin: phylad A is practically the only phylad in Black Sea, whereas its frequency drops to 0.85 to the northern Aegean, to 0.40 in the rest of Mediterranean and the Bay of Biscay, and to 0.14 in northern Adriatic. This pattern strongly suggests that the gene flow between the Black Sea and the Mediterranean is unidirectional from the former into the latter. Gene barriers of a lesser effect exist between northern and southern Aegean and between the Adriatic and the rest of Mediterranean. These observations are of fundamental importance on the understanding recruitment patterns and on management of the species in the Mediterranean, because they illustrate an asymmetry in dependence of one stock on the other and also emphasise the importance that historical events, such as glaciation and present hydrographic forces, have on the distribution and genetic mixing of populations of harvested fish.

The present project aims to provide direct evidence about the causes of the pattern of population differentiation described above by:
a) examining the mtDNA variation in more samples of adult individuals from critical geographical areas,
b) extending the analysis to larvae by using PCR-based techniques, since under the established model larval drift is thought to play a pivot role in the formation of the observed population structure,
c) proceeding to sequencing analysis of the control region of mtDNA, in order to increase the resolution power of the genetic study and to estimate more accurately the relative evolutionary distance between the haplotypes revealed by RFLP analysis,
d) developing a quick assay for phylad characterisation, in order to technically simplify scoring of the individual animals regarding their phylad, thus facilitating future work for routine stock monitoring, and
e) introducing in the analysis a highly polymorphic nuclear marker, microsatellite DNA, in order to increase the resolution power of the analysis and to test the hypothesis that the Black Sea population may be a different species or subspecies of anchovy.

Coordinator

INSTITUTE OF MARINE BIOLOGY OF CRETE
Address
Main Port Heraklion
Heraklion
Greece

Participants (2)

UNIVERSIDADE DO ALGARVE
Portugal
Address
322,Campus De Gambelas
8000 Faro
UNIVERSITA DEGLI STUDI DI PADOVA
Italy
Address
Via U.bassi 58/B
35131 Padova