In the Marine Fish Atherinella Brasiliensis (Teleostei

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 Politećnico, 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; 3Nućleo de Pesquisas em agua´ Bay (Parana´ State) and one from the Conceiçaõ Lagoon (Santa Catarina Limnologia, Ictiologia e Aquïcultura (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 Conceiçaõ 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 Conceiçaõ 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 specimens 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 Politećnico, 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). (França 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

2009 The Authors 242 Journal compilation 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 Conceiçaõ 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 Conceiçaõ 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 Conceiçaõ Lagoon (Santa Catarina Parana´ State; in Pontal do Parana´,attheentranceofParanag- State) the random amplified polymorphic DNAs (RAPDs) ua´ Bay (2534¢ S, 4821¢ W), 30 specimens were collected, were chosen because of their wide use in population genetics and 29 were obtained in Laranjeiras Bay (2535¢ S/ and the availability of documentation for many different 4819¢ W), which is connected to the Paranagua´ Bay. organisms, including fish (Dergam et al. 1998). This tech- Another 30 specimens were collected in ConceiçaõLagoon nique allows the detection of a large number of polymor- (2735¢ S, 4815¢ 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 through phenol/chloroform (1 : 1, v : v) and then chloroform, after mutagenic activity, or indirectly via population-mediated which it was precipitated with saline solution (NaCl 5 M)and processes (i.e. selection, bottlenecks). The RAPDs can be 100% cold ethanol followed by an overnight incubation at

2009 The Authors Journal compilation 2009 The Royal Swedish Academy of Sciences 243 Genetic population structure Atherinella brasiliensis • da Silva Cortinhas et al. Acta Zoologica (Stockholm) 91: 242–248 (April 2010)

Fig. 1—Location where Atherinella brasiliensis was collected: 1, Conceic¸a˜o Lagoon (SC); 2, Pontal do Parana´ (PR);3,LaranjeirasBay(PR).

–20 C. The pellet was re-suspended in 60 lL1/10TEbuffer 1.4% agarose gel submerged in 1 · TBE buffer for 4 h at (Tris–HCl 10 mM pH 7.5, EDTA 1 mM pH 8.0) containing 60 V. Afterwards, the products were stained with ethidium RNAse 10 mg/mL and incubated in a water-bath at 37 Cfor bromide (0.05%), visualized under ultraviolet light and 90 min. The quantity of DNA in each sample was estimated photographed. Each band observed in the gel was considered through the comparison with the phage k DNA, for which the a Mendelian segregation locus and a binomial matrix (1/0) concentration was previously calculated using electrophoresis was generated based on presence/absence of bands, in 0.8% agarose gel for 90 min at 100 V. Afterwards, the respectively, with the aim of calculating the intra- and inter- DNA was stained with ethidium bromide (0.05%), visualized populational diversities. under ultraviolet light and photographed using an Olympus The similarity matrices were generated through the NTSYS camera. software version 2.1 (Rohlf 2000), adopting the Jaccard (1901) coefficient which plotted a principal coordinate analysis (PCO) to verify the dispersion of the collected individuals. RAPD and data analysis Using the same software, the cophenetic correlation was per- After the tests to accurately determine the concentration of formed to compare the two symmetric matrices, and grouping the main reagents of the polymerase chain reaction were per- analyses were obtained by the UPGMA method (Sneath and formed, a band pattern was obtained for each primer and the Sokal 1973). Bootstrap values for the UPGMA method were reactions were repeated several times to ensure the reliability obtained through the BOOD software version 3.03 (Coelho of the results. 2004). To confirm the results obtained by the UPGMA method, The amplification of the RAPD bands was performed grouping analyses through the neighbour-joining method according to the Bardakci and Skibinski (1994) conditions, were also performed using the software FREETREE (Pavlicek with a reaction mixture of 13 lL total volume containing: et al. 1999). The Mantel test (1967) was used through the 10 ng DNA, 0.46 lM primer, 1 U Taq polymerase, 2 mM MANTEL STRUCT 1.0 software (Miller 1999) to evaluate the

MgCl2,200lM dNTP, Tris–KCl buffer (Tris–HCl 20 mM genetic differentiation among the specimens of A. brasiliensis, pH 8.0 and KCl 50 mM) and ultra-pure water enough to through the comparison between the similarity and the model complete the volume. The Techne thermocycler was used matrix that expresses the origin of the individuals from pair- and programmed according to the conditions proposed by Al- wise comparisons between the groups. meida (1998), with an initial stage of 4 min at 92 C, followed A genetic distance Euclidean matrix was used to generate by 40 cycles of 1 min at 92 C, 90 s at 40 Cand2minat an analysis of molecular variance (AMOVA) according to 72 C. The ﬁnal stage was 72 C for 5 min, followed by cool- Excofﬁer et al. (1992) through the ARLEQUIN software version ing at 20 Cfor20minand4Cfor30min. 2000 (Schneider et al. 2000) to estimate a possible genetic Twenty-fourprimersoftheOPA,OPE,OPK,OPXand structuring among the individuals from the places studied.

OPW (Operon Technologies Inc., Alameda, CA, USA) kits This analysis provides the hST, which is analogous to the were tested for the oligonucleotide selection used in the pres- Wright (1978) FST, which represents one measure of the ent report, but only 12 were chosen based on repeatability, genetic differentiation degree or interpopulational sub- band clear pattern and presence of polymorphic bands. The division (Slatkin 1993). According to Wright (1978), to obtain ampliﬁcation products were submitted to electrophoresis in a signiﬁcant estimative different from zero, results between 0

2009 The Authors 244 Journal compilation 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

Table 1 Random amplified polymorphic DNA (RAPD) primers, Both the phenogram obtained through the neighbour-join- corresponding to sequences and number of bands associated with ing method and the one generated through the UPGMA each primer method (not shown) presented low bootstrap values. Besides, the phenogram obtained through the UPGMA method Number of bands presented a low cophenetic correlation (0.69), suggesting a short correspondence between the generated matrices Primer Sequence (5¢–3¢) n n t p through the phenogram and the matrix obtained through the OPW03 GTCCGGAGTG 5 4 observations. We can therefore infer that the generated phen- OPW04 CAGAAGCGGA 8 6 ograms were not the best representation of the genetic diver- OPW06 AGGCCCGATG 7 3 sity pattern among the specimens of the different places OPW09 GTGACCGAGT 7 5 studied. OPW17 GTCCTGGGTT 8 5 ThePCO,basedontheJaccardsimilaritymatrix,wasthe OPX04 CCGCTACCGA 13 11 best indicator of genetic diversity among the studied individu- OPX07 GAGCGAGGCT 6 4 OPX17 GACAGGGACC 10 9 als (Fig. 3). Through the PCO it was possible to observe that OPX20 CCCAGCTAGA 3 2 the specimens from Pontal do Parana´ and Laranjeiras (Parana´ OPA06 GGTCCCTGAC 5 3 State) comprised a homogeneous population. This hypothesis OPE09 CTTCACCCGA 3 2 wasconfirmedbytheAMOVA results and by the Mantel test OPK14 CCCGCTACAC 4 1 (Tables 2 and 3), with significant P-values. Because these two Total 79 55 regions are very close, gene flow between the individuals of both places is likely. The total number of scorable bands and the number of polymorphic fragments are represented by nt and np, respectively. Comparing the specimens from ConceiçaõLagoon(Santa Catarina State) and from Parana´ State, there was an increase in the genetic diversity (r ¼ 0.1627 and r ¼ 0.1530, respectively) observed through the Mantel test (Table 3), and also and 0.05 indicate low genetic structuring; between 0.05 and in the genetic structuring (hST ¼ 0.07519 and hST ¼ 0.25 indicate moderate genetic structuring; and over 0.25 0.07432, respectively) observed through AMOVA (Table 2), indicate a high genetic structuring. with highly significant P. According to Wright (1978) it is possible to deduce the occurrence of incipient genetic structuring among the individuals of this area compared with others previ- Results ously analysed. However, the results are still too few to geneti- Seventy-nine bands with variable polymorphism between cally differentiate the specimens of the three places, so they 2000 and 300 base pairs (bp) (Table 1) were obtained, can be considered as a unique population. which indicates a high intrapopulational genetic variation compared with the interpopulational relations (Table 2). Discussion Figure 2 shows the electrophoretic profile obtained with the OPX07 primer and the polymorphic band variation is indi- The interpopulational genetic variation levels of marine fish cated by the arrow (~600 bp). This figure corroborates the are, in theory, lower than those of freshwater fish. This finding results observed for other primers, which showed that the seems to be associated with the population structures of the variability occurred predominantly within the areas studied marine species, which present a less restricted gene flow; and not between them. also, the formation of geographical barriers is much less

Table 2 Analysis of molecular variance (AMOVA –Excofﬁeret al., 1992) with P values obtained from 1023 permutations

Variation

Locations Source Percentage hST P-value

Pontal do Parana´ (PR) · Laranjeiras (PR) Among loc. 2.02 0.02016 0.01173 Within loc. 97.98 Pontal do Parana´ (PR) · Conceiçaõ Lagoon (SC) Among loc. 7.52 0.07519 0.00000 Within loc. 92.48 Laranjeiras (PR) · Conceiçaõ Lagoon (SC) Among loc 7.43 0.07432 0.00000 Within loc. 92.57

Loc., localities.

2009 The Authors Journal compilation 2009 The Royal Swedish Academy of Sciences 245 Genetic population structure Atherinella brasiliensis • da Silva Cortinhas et al. Acta Zoologica (Stockholm) 91: 242–248 (April 2010)

Fig. 2—Profile of random amplified polymorphic DNA electrophoresis of Atherinella brasiliensis from Pontal do Parana´ (Parana´ State), Laranjeiras (Parana´ State) and Conceiçaõ Lagoon (Santa Catarina State) showing the amplified fragments by primer OPX 07. The first and the last columns contain the molecular weight marker fragments (a ladder of 100 base pairs, PB-L Productos Bio-Lo´gicos), presenting a first additional band of 1434 base pairs. Lanes 1 to 10, the specimens from Pontal do Parana´ (Parana´ State); lanes 11 to 19, the specimens from Laranjeiras (Parana´ State); and lanes 20 to 29, the specimens from Conceiçaõ Lagoon (Santa Catarina State).

common than in continental waters (Ward et al. 1994). How- ever, with the increasing number of studies using molecular markers in marine fish, this observation may not be a general rule and the genetic structure of these populations may depend on several other factors such as ecology, demography, behaviour, genetics, oceanography, climate and tectonism (Affonso 2004). In the samples of the Pontal do Parana´ (Parana´ State) area, variations in the intensity, loss or gain of bands were not detected among the 79 analysed RAPD markers compared with fish of other areas. This finding suggests that ecological factors (i.e. polluted environment) did not interfere in the alteration of the genetic variability in the fish from this area. The 55 polymorphic loci occurred in the individuals from the three areas showing high intrapopulational variability and a low interpopulational variability. The results of the present report are in accordance with other cytogenetic data available in the literature (Sola et al. 1988; Sczepanski et al. 2007), in Fig. 3—Principal coordinate analysis based on Jaccard genetic which the karyotypic diversity in Atheriniformes is related to similarity (1901). The specimens from Pontal do Parana´ (Parana´ heteromorphisms within some pairs explaining the differences State) are represented in white; the specimens from Laranjeiras in the fundamental number. (Parana´ State) are in grey and the specimens from ConceiçaõLagoon Fish, in common with most animals, often show a (Santa Catarina State) are in black. subdivision of each species into local populations which are isolated to varying degrees. Among freshwaters, populations inhabiting isolated lakes or rivers with physical barriers Table 3 Mantel test (1967) between are reasonably easy to define, whereas populations of marine fish are usually less clearly delineated. If localized Populations rPR2 populations inhabit similar environments or remain interconnected through migration and gene flow, they may Pontal do Sul (PR) x Laranjeiras (PR) 0.0394 0.0188 0.0016 display largely homogeneous arrays of phenotypic or genetic Pontal do Sul (PR) x Lagoa da Conceiçaõ (SC) 0.1627 0.0001 0.0265 traits (Carvalho 1993). This fact can be observed in the Laranjeiras (PR) x Lagoa da Conceiçaõ (SC) 0.1530 0.0001 0.0234 samples of Pontal do Sul and Laranjeiras Bay (Parana´ State) Correlations (r), respective Type I (P) and determination coefficients (R2), where the gene flow is intense and, because of the proximity obtained in with 10.000 random permutations. of these areas, they tend to become homogeneous and, as a

2009 The Authors 246 Journal compilation 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 result, they follow similar evolutionary paths. Regarding the References samples of Conceiçaõ Lagoon, where the gene flow is Affonso, P. R. A. M. 2004. Marcadores moleculares na ana´lise de espećies restricted, incipient structuring could be explained probably ecomposiçaõ populacional de peixes marinhos de recifes de corais da by the environmental similarity that selects individuals with Famı´lia Pomacanthidae (Perciformes). Tese (Doutorado em Gene´tica similar characteristics. eEvoluçaõ). Centro de Cieˆncias Biolo´gicas e da Sau´de, Universid- Our data concerning RAPD markers indicate three possible ade Federal de Saõ Carlos, 158 pp. hypotheses: Almeida, F. S. 1998. Ana´lise da variabilidade gene´tica em Pimelodidae e (i) According to Chapman et al. 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2009 The Authors 248 Journal compilation 2009 The Royal Swedish Academy of Sciences