Acta Zoologica (Stockholm) 91: 242–248 (April 2010) doi: 10.1111/j.1463-6395.2009.00404.x A prime inference on genetic diversity (RAPDs) in the marine fish Atherinella brasiliensis (Teleostei, Atherinopsidae) from Southern Brazil Maria Cristina da Silva Cortinhas1, Chirlei Glienke1,AlbertoJose´ Prioli2,3, Rafael Bueno Noleto1, Daniele Aparecida Matoso1 and Marta Margarete Cestari1 Abstract 1Laborato´rio de Citogene´tica Animal, DaSilvaCortinhas,M.C.,Glienke,C.,Prioli,A.J.,Noleto,R.B.,Matoso, Departamento de Gene´tica, Setor de D. A. and Cestari, M. M. 2010. A prime inference on genetic diversity Cieˆncias Biolo´gicas, Universidade Federal (RAPDs) in the marine fish Atherinella brasiliensis (Teleostei, Atherinopsidae) do Parana´, Centro Polite´cnico, Curitiba, from Southern Brazil. — Acta Zoologica (Stockholm) 91: 242–248 Parana´, Brazil, CEP 81531-990; 2Departamento de Biologia Celular e As a result of the importance of Atherinella brasiliensis in estuarine environ- Gene´tica, Universidade Estadual de ments, random amplified polymorphic DNA (RAPD) markers were used to Maringa´,Maringa´,Parana´, Brazil, CEP verify the genetic diversity in A. brasiliensis from two different places in Paran- 87020-900; 3Nu´cleo de Pesquisas em agua´ Bay (Parana´ State) and one from the Conceic¸a˜o Lagoon (Santa Catarina Limnologia, Ictiologia e Aqu¨icultura (NUPELIA), Universidade Estadual de State). Cytogenetic data have shown a high karyotypic diversity in some popu- Maringa´,Maringa´,Parana´, Brazil, lations, although in others this peculiarity demonstrates rearrangements such as 87020-900 heterochromatinization. In the present study, a low level of genetic structuring between the samples from Conceic¸a˜o Lagoon compared with the others was Keywords: observed through principal coordinate analysis (PCO), analysis of molecular Atherinella brasiliensis, silversides, genetic variance and Mantel test according to 79 RAPD markers. As this specie does variability, conservation genetics, RAPD not perform horizontal migration and the individuals of Conceic¸a˜o Lagoon are isolated, three hypotheses are proposed to explain the results: (i) similar envi- Accepted for publication: ronments may show homogeneous populations not depending on the geo- 17 February 2009 graphical distance, (ii) because vicariant events that formed the bays occurred in a recent period, the fragmentation effects over the structuring of the genetic diversity may still be low and not totally detectable by the RAPD technique and (iii) the isolation time or the number of generations may not be enough to promote a possible differentiation and genetic structuring between the speci- mens of these three places. The specimens of these places present a low level of differentiation and genetic structuring so we can consider them as a unique homogeneous population. MariaCristinadaSilvaCortinhas,Laborato´rio de Citogene´tica Animal, Departamento de Gene´tica, Setor de Cieˆncias Biolo´gicas, Universidade Federal do Parana´, Centro Polite´cnico, Jardim das Ame´ricas, Curitiba, Parana´, Brazil, CEP 81531-990. E-mail: [email protected] In Brazil, the king-fish of the Atherinopsidae family are Introduction represented by the Odontesthes and Atherinella genera. Within The order Atheriniformes is a monophyletic group known as the Atherinella genus, Atherinella brasiliensis (Quoy and silversides or king-fish, and it is composed of six families Gaimard 1824) can be found from Pernambuco State (among them Atherinopsidae) and 49 genera (Dyer 2006). (Franc¸a 2005) to Rio Grande do Sul State (Bemvenuti These fish are distributed in marine, fresh and estuarine 1987), mainly in coastal waters and especially in river waters and in tropical and temperate environments all around mouths and brackish water regions (Figueiredo and Menezes the world (Beheregaray and Sunnucks 2001). 1978).Thisfishhasbeenwidelyusedbothasasourceof Ó 2009 The Authors 242 Journal compilation Ó 2009 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 91: 242–248 (April 2010) da Silva Cortinhas et al. • Genetic population structure Atherinella brasiliensis complementary wealth and even as a food (Hostim-Silva et al. used as biomarkers of loss or gain of genetic diversity in 1995) because it is a resident estuarine species (Pessanha organisms that live in contaminated environments (Wolf et al. and Arau´jo 2001) and is constant and abundant in these 2004), and are also useful in genotoxicity and carcinogenesis environments (Paiva Filho and Gianinni 1990; Pessanha et al. studies (Atienzar and Jha 2006). The threat to A. brasiliensis is 2000; Vendel et al. 2000). of great concern because the biological diversity involves not Da Silva Cortinhas et al. (2003) performed a cytogenetic only the variety of species, but also the intraspecific genetic study in A. brasiliensis from the Paranagua´ Bay (Parana´ State) variation. and Conceic¸a˜o Lagoon (Santa Catarina State) detecting Atherinella brasiliensis does not perform horizontal migration intra- and interpopulational chromosomal variation, probably (Hostim-Silva et al. 1995) living only in estuary environments. determined by pericentric inversions. Most of the analysed This feature represents a barrier to gene flow, increasing the individuals presented a diploid number of 48 chromosomes chances that genetic differences may accumulate with time. It constituted by four metacentric, 10 submetacentric, 14 sub- is relevant to point out that both contaminated environments telocentric and 20 acrocentric chromosomes. The pattern and non-horizontal migration may act as independent factors karyotype had a fundamental number equal to 76. However, that might promote differentiation and a possible genetic five different cytotypes were found in the specimens from structuring among fish from these areas. Paranagua´ Bay (Parana´ State) and only one different cytotype Considering the possible isolation of A. brasiliensis popula- in the specimens from the Conceic¸a˜o Lagoon (Santa Catarina tions because of the lack of horizontal migration, and also the State). contamination observed in the areas inhabited by this species, According to Sczepanski et al. (2007), variations in hetero- the diagnosis and interpretation of the genetic variation in this chromatinization of short arms may produce hetero- species is extremely important. This knowledge will support morphisms within some pairs, explaining the differences in protection and conservation programmes for the maintenance the fundamental number in this group, although for Sola of the diversity of this species. et al. (1988) this feature in Atherinopsidae species is partially related to the activity of nucleolar organizing Materials and Methods region (NORs) as well as to structural differences in hetero- chromatic regions. Sample collection For a better understanding of the genetic diversity of A. brasiliensis from Pontal do Parana´ and Laranjeiras Bay Atherinella brasiliensis was collected in two different places in (Parana´ State) and Conceic¸a˜o Lagoon (Santa Catarina Parana´ State; in Pontal do Parana´,attheentranceofParanag- State) the random amplified polymorphic DNAs (RAPDs) ua´ Bay (25°34¢ S, 48°21¢ W), 30 specimens were collected, were chosen because of their wide use in population genetics and 29 were obtained in Laranjeiras Bay (25°35¢ S/ and the availability of documentation for many different 48°19¢ W), which is connected to the Paranagua´ Bay. organisms, including fish (Dergam et al. 1998). This tech- Another 30 specimens were collected in Conceic¸a˜oLagoon nique allows the detection of a large number of polymor- (27°35¢ S, 48°15¢ W), city of Floriano´polis, Santa Catarina phismsinDNAsegmentsdistributed throughout the genome State (Fig. 1). of the studied individuals, mainly because of the quantity of A sample of approximately 100 mg of the dorsal muscle available primers used to detect variations in some regions was removed from each sample. The muscular tissue was of the genome (Bardakci and Skibinski 1994). This analysis stored in ethanol 96% and preserved in a freezer at –20 °C. may be more sensitive than traditional methods because it involves non-coding neutral regions, which allow high muta- DNA extraction and purification tion levels (Cagigas et al. 1999). This marker has been very useful in the management of neotropical fish in places The total DNA was extracted by the phenol/chloroform affected by human activities, relating genetic variability data method according to Sambrook et al. (1989) with some modi- with environmental parameters (Hatanaka and Galetti fications. The sample of the muscle was macerated in liquid 2003; Matoso et al. 2004; Sofia et al. 2006) and identifying nitrogen and homogenized in 500 lLPSbuffer[Tris–HCl population structuring (Prioli et al. 2002; Almeida et al. 0.2 M pH 9.0, ethylenediaminetetraacetic acid (EDTA) 2003; Leuzzi et al. 2004; Torres and Ribeiro 2009). 30 mM pH 8.0, sodium dodecyl sulphate 2% and saccharose The contamination of some areas where A. brasiliensis is 5%], 500 lLTHbuffer(Tris–HCl10mM pH 7.5, NaCl found is harming the populations, and loss of genetic varia- 60 mM,EDTA10mM pH 8.0, saccharose 5%, spermine tion, as observed in Paranagua´ Bay (Parana´ State) near 0.15 mM and spermidine 0.15 mM)and6lL K proteinase Paranagua´ Seaport, is one of the problems. According to (20 mg/mL) and incubated in a water-bath for 90 min at Wolf et al. (2004), toxic compounds may interfere in the 50 °C. Afterwards, the DNA was purified by extraction with genetic constitution of populations, either directly