SCI. MAR., 67 (1): 43-52 SCIENTIA MARINA 2003 Genetic differentiation of Solea solea (Linnaeus, 1758) and Solea senegalensis Kaup, 1858, (Pisces: Pleuronectiformes) from several estuarine systems of the Portuguese coast* HENRIQUE N. CABRAL1, FILIPE CASTRO2, DIANA LINHARES2 and PAULO ALEXANDRINO2,3 1 Instituto de Oceanografia / Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade de Lisboa, Rua Ernesto de Vasconcelos, 1749-016 Lisboa, Portugal. E-mail: [email protected] 2 Centro de Estudos de Ciência Animal, ICETA Universidade do Porto, Campus Agrário de Vairão, 4485-661 Vairão, Portugal. 3 Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do Porto, Praça Gomes Teixeira, 4099-002 Porto, Portugal. SUMMARY: The genetic differentiation of Solea solea and Solea senegalensis from several estuarine systems along the Portuguese coast was studied. Nine polymorphic isozyme loci (ACP-1*, ACP-2*, GPI-1*, GPI-2*, sMDH*, ME-1*, ME- 2*, MPI* and PGM*) were analysed using starch gel electrophoresis and isoelectric focusing. Differentiation between the species was high (mean average Nei distance of 0.93). The most efficient loci in the diagnosis of the two species were ACP- 1*, ME-2* and GPI-2*. S. solea showed a higher genetic diversity than S. senegalensis. Within each species a low genetic differentiation between the samples analysed was found. Although with a low magnitude the interpopulational genetic dif- ferentiation of S. solea was higher than that of S. senegalensis. This could probably be explained by some particularities of the life cycles of these species, namely the more extended period of occurrence of larval stages of S. senegalensis in the plankton. Although no clear evidence about the population structure model emerged from the analysis of several Atlantic and Mediterranean populations of S. solea, the significant correlations obtained between genetic and geographical distances support an isolation by distance model. Key words: genetic structure, population genetics, allozyme, Solea, Portugal. RESUMEN: DIFERENCIACIÓN GENÉTICA DE SOLEA SOLEA (LINNEUS, 1758) Y SOLEA SENEGALENSIS KAUP, 1858), (PISCES: PLEU- RONECTIFORMES) EN VARIOS SISTEMAS ESTUÁRICOS DE LA COSTA PORTUGUESA. – Se estudió la diferenciación genética de Solea solea y Solea senegalensis en varios sistemas estuáricos a lo largo de la costa de Portugal. Se analizaron nueve loci de iso- zimas polimórficos (ACP-1*, ACP-2*, GPI-1*, GPI-2*, sMDH*, ME-1*, ME-2*, MPI* and PGM*) usando electroforesis en gel de almidón y enfoque isoeléctrico. La diferenciación entre especies fue alta (media de la distancia Nei 0.93). El loci más eficiente en la diagnosis de las dos especies fue ACP-1*, ME-2* and GPI-2*. S. solea presentó una mayor diversidad genética que S. senegalensis. Se encontró una baja diferenciación genética entre muestras analizadas dentro de una misma especie. Aunque con una baja magnitud, la diferenciación genética entre poblaciones de S. solea fue más alta que la de S. senegalensis. Esto podría explicarse, probablemente, por algunas particularidades de los ciclos de vida de estas especies, como el más prolongado periodo de aparición de los estadios larvarios de S. senegalensis en el plancton. Aunque no hay una evidencia clara sobre el modelo de estructura de la población de S. solea, las correlaciones significativas obtenidas apoyan un aislamiento por un modelo de distancia. Palabras clave: estructura genética, genética de poblaciones, allozymas, Solea, Portugal. *Received May 28, 2001. Accepted September 9, 2002. GENETIC DIFFERENTIATION OF S. SOLEA AND S. SENEGALENSIS 43 INTRODUCTION ers to larval dispersion. Other features might, how- ever, result in “large scale” genetic exchanges, con- Estuarine fish communities comprise species ferred by the long duration of the larval period and a with different life-history patterns. A particularly high individual fecundity. important component of these communities is repre- The present work aims to study the genetic dif- sented by marine species that use estuarine systems ferentiation between S. solea and S. senegalensis as nursery areas, in order to benefit from the high along the Portuguese coast based on isozyme analy- food availability and the existence of few predators sis. This geographical area is particularly of interest (Haedrich, 1983; McLusky, 1989). since almost no other studies have been done and European sole, Solea solea (Linnaeus, 1758) and this is the principal zone in the Atlantic where these Senegalese sole, Solea senegalensis Kaup, 1858, are species are sympatric. Furthermore, the geomorpho- marine species with this kind of life cycle. These logical particularities of the Portuguease coast, e.g. two flatfish species have a very similar morphology a narrow continental shelf divided by deep canyons, and a sympatric distribution from North Africa and could induce a different population substructuring the western Mediterranean up to the Bay of Biscay pattern compared to north European areas, which (Quéro et al., 1986). While ecological information are much shallower. The results obtained for the concerning S. senegalensis is scarce and refers Portuguese coast may also provide a better under- almost exclusively to the Portuguese coast (e.g. standing of the genetic differentiation of these Andrade, 1989; Costa and Bruxelas, 1989; Cabral species a broader geographical scale, especially with and Costa, 1999), substantial knowledge has been regard to the differences between north European gained on S. solea, mainly in north European estuar- and Mediterranean populations reported by several ies and coastal areas. The adult populations of S. authors (e.g. Koutolas et al., 1995; Exadactylos et solea are located on the continental shelf, where al., 1998). spawning takes place at water depths ranging from 40 to 100 m (Koutsikopoulos et al., 1989; Kout- sikopoulos and Lacroix, 1992). Larvae and juveniles MATERIAL AND METHODS tend to migrate to coastal areas by both passive and active transport processes that are not yet complete- Samples were collected between July and August ly understood (Marchand and Masson, 1989, Kout- 1997 in four estuarine systems of the Portuguese sikopoulos and Lacroix 1992; Amara et al., 1994, coast: Ria de Aveiro, Tagus and Mira estuaries and 1998). Juveniles of S. solea concentrate in estuaries Ria Formosa (Fig. 1). Beam trawls and trap nets and in bays for a period of about two years, after were used to catch the fish. All specimens caught which they start to migrate off coastal areas (Kout- were less than two years old. Due to their differen- sikopoulos et al., 1989). tial pattern of abundance, few individuals of S. sene- As for other marine fish species, the main feature galensis were captured in Mira estuary (and thus of the life cycle of S. solea and S. senegalensis is a they were not considered for the genetic analysis) division into a juvenile phase that is predominantly and none of S. solea in Ria Formosa. estuarine and an adult phase that is marine. This life- Liver, muscle and blood tissue samples were history pattern may have an impact on the structur- taken immediately after capture and stored at –80ºC ing of offshore adult populations, particularly on the until electrophoretic analysis. Liver and muscle cells genetic differentiation, since a strong association were disrupted by use of ultrasound after adding dis- between a particular spawning and nursery area can tilled water. The tissue enzymes were analysed by be expected. horizontal starch gel electrophoresis (SGE) or iso- Although several studies on the genetics of Solea electric focusing (IEF). IEF was used since for some spp. have been carried out (e.g. Quignard et al., of the markers the interpretations of the patterns in 1984; Pasteur et al., 1985; Goucha et al., 1987; She the gels were not satisfactory when SGE was used. et al., 1987), only a few were focused on population The technical procedures of these methods are genetics (e.g. Koutolas et al., 1995; Exadactylos et described in Murphy et al. (1996) and Alexandrino al., 1998). According to Koutolas et al. (1995), et al. (1996). Histochemical staining methods were some features of the life cycle of S. solea may adapted from Harris and Hopkinson (1976). Among induce a low genetic flux; these include the homing the 24 loci examined (Table 1), only a sub-set of behaviour to spawning grounds and physical barri- nine loci was used in the analysis, since when a cer- 44 H.N. CABRAL et al. 30°W 20°W 10°W 0° 10°E 20°E 30°E 40°E 50°N 1 3 2 45 6 7 8 2118 40°N 19 1213 22 11 14 20 1025 26 9 23 16 15 27 30°N 17 20°N 24 10°W 0° 10°E 20°E 30°E FIG. 1. – Atlantic and Mediterranean samples for which the data on the common genetic markers were analysed (Samples 1 to 17: S. solea, Koutolas et al. (1995) data; 18 to 20: S. solea, this study (18: Ria de Aveiro; 19: Tagus estuary; 20: Mira estuary); 21 to 23: S. senegalensis, this study (21: Ria de Aveiro; 22: Tagus estuary; 23: Ria Formosa); 24 and 25: S. senegalensis, Goucha et al. (1987) data; 26 and 27: S. aegyptiaca, Goucha et al. (1987) data). TABLE 1. – Enzyme systems, loci studied, separation technique, buffer system and tissue. Protein E. C.1 Locus 1, 2 Sep. tec./ Buf. syst.3 Tissue 4 Aspartate aminotransferase 2.6.1.1 sAAT-1* (a) sAAT-2* (a) SGE/C, D, E L, M sAAT-3* (a) Acid phosphatase 3.1.3.2 ACP-1* SGE/B L ACP-2* IEF Adenylate kinase 2.7.4.3 AK* (a) SGE /D L, M Creatine kinase 2.7.3.2 CK* (a) SGE /D L, M Esterase 3.1.1.- EST-1* (a) EST-2* (a) SGE/C L, M EST-3* (a) Glycerol-3-phosphate dehydrogenase 1.1.1.8 G3PDH* (b) SGE/C L, M Glucose-6-phosphate isomerase 5.3.1.9 GPI-1* SGE/A M GPI-2* Hemoglobine - HB* (b) IEF B L-Iditol-dehydrogenase 1.1.1.14 sIDDH* (b) SGE/D L, M L-Lactate dehydrogenase 1.1.1.27 LDH-2* (b) SGE/A L, M LDH-3* (b) Malate dehydrogenase 1.1.1.37 sMDH* SGE/C M Malic enzyme (NADP+) 1.1.1.40 ME-1* SGE/C M ME-2* Mannose-6-phosphate isomerase 5.3.1.8 MPI* SGE/C L Phosphogluconate dehydrogenase 1.1.1.44 PGDH* (b) SGE/C L, M Phosphoglucomutase 5.4.2.2 PGM* SGE/A M Superoxide dismutase 1.15.1.1 SOD-1* (b) SGE/A L, M 1 according to Shaklee et al.