Material and methods. Samples

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4 290 Material and methods Samples Six strains, derived from single females collected in the wild, from different D. gouveai population were analyzed: J79L67 (Ibotirama/BA); J18E2 (Pireno polis/go); H27S1 (Peixoto/MG); H74M2 (Altino polis/sp); J82F3 and J83F3 (Analândia/ SP). Their geographical localization is presented in Figure 2. The Ibotirama locality belongs to Caatinga Domain, where there are many cactaceae species. The other localities studied are sandstone table hills, such as Analaˆ ndia/sp (Figure 1), with Pilosocereus vilaboensis (Pireno polis/go) or Pilosocereus machrisii (all another localities) growing in the rock fields of hilltops. Isolation of pbum-2 satellite DNA and sequence analysis The genomic DNA of the Drosophila specimens was extracted according to Preiss, Hartley and Artavanis-Tsakonas (1988). To isolate the copies of the pbum-2 family, reactions of PCR were carried out using the specific primers P1 (5 -CGG AGT ATT TTT CAT TCG AC-3 ) and P2 (5 - GGT ATG CCA TAA AGA AGT CG-3 ). The resultant bands of approximately 400 bp were eluted from the gel by overnight incubation in an elution solution (500 mm NaAc; 1 mm EDTA). The recovered fragments were cloned using the pmosblue blunt ended cloning kit RPN 5110 (Amersham Pharmacia Biotech). The recombinant clones were identified by the blue-white system of the gene from b-glycosidase (Sambrook, Fritsch & Maniatis, 1999). The DNA template reaction for sequencing was prepared according to the BigDye Terminator Cycle Sequencing Ready Reaction Kit manual (Perkin-Elmer). Automatic DNA sequencing was performed on an ABI Prism TM 377 sequencer (Perkin-Elmer). The alignment of the sequences was carried out in the CLUSTAL W (version 1.8, Thompson, Higgins & Gibson, 1994). The genetic distances were calculated according to the Kimura 2 parameters model (Kimura, 1980) using the MEGA 3.0 program (Kumar, Tamura & Nei 2004). The distance matrix was used to make a dendrogram using the Neighbor-Joining method (Saitou & Nei, 1987). Analysis of molecular variance (AMOVA) of the pbum-2 satellite DNA family from D. gouveai was performed using the ARLEQUIN software (Schneider et al., 2000), according to the method described in Excoffier, Smouse and Quattro (1992). To compare the values found for D. gouveai, a AMOVA with the pbum-1 sequences from D. buzzatii (Kuhn et al., 2003) and the pbum-2 sequences from D. seriema (Kuhn & Sene, 2004) was performed. Results and discussion Figure 2. Drosophila gouveai collection sites in Brazil. (1) Ibotirama/BA (12 16 S; W) (J79L67 strain). (2) Pireno polis/ GO (15 48 S; W J18E2 strain). (3) Peixoto/MG (20 23 S; W H27S1 strain). (4) Furnas/MG (20 37 S; W H24S1 strain). (5) Altinópolis/SP (21 48 S; W H74E2 strain). (6) Analândia/SP (22 08 S; W J82F3 and J83F2 strains). BA, Bahia state. GO, Goiás state. MG, Minas Gerais state. SP, São Paulo state. Twenty eight pbum-2 satellite DNA monomers were obtained: seven from locality (1) (GenBank accession nos. AY AY953030), four from locality (2) (GenBank accession nos. AY AY953034), two from locality (3) (GenBank accession nos. AY AY953051), four from locality (4) (GenBank accession nos. AY AY953049) and 11 from locality (6) (GenBank accession nos. AY AY953045). These sequences were analyzed together with eight previously described pbum-2 sequences from locality (4) (Kuhn & Sene, 2005) (GenBank accession nos. AY AY656608). The high nucleotide similarity among the sequences obtained in this

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6 ), no repetition unit of the pbum-2 satellite DNA characteristic of D. antonietae was observed in the population of Analaˆ ndia/sp locality. This could be related to the observations that mitochondrial genes tend to introgress more easily than the nuclear genes (Dorado, Rieseberg & Arias, 1992; Arnold, 1993). The most cited explanations for this observation are related to cytonuclear disequilibria processes (Arnold, 1993) and sterility of hybrid males (Aubert & Solignac, 1990). The values of genetic distance, based on the Kimura 2 parameter model (Kimura, 1980), among all the pbum-2 sequences analyzed from the six D. gouveai populations showed high level of similarity (with values ranging from 0 to 0.045, with an average of Table 1). Moreover, the average genetic distance in each population reached as maximum value, showing that the intrapopulational variation is similar to the interpopulational variation (Table 1). Furthermore, there was no occurrence of an assembly of all sequences from the same population in the same branch in a NJ dendrogram (Figure 4), indicating that the pbum-2 is highly conserved among the D. gouveai populations. The AMOVA of pbum satellite DNA sequences was performed considering each population as a group, showing that 100% of the total genetic diversity could be ascribed to intrapopulational variability in D. gouveai, with no interpopulational differentiation. The Fct (fixation index among groups) calculated was low (0.006) and without statistical support (Table 2). Previous studies revealed a high degree of similarity of pbum-1 satellite sequences among 13 D. buzzatii populations in South America (Kuhn et al., 2003) and pbum-2 satellite sequences Table 1. Intrapopulational and total genetic distance calculated for pbum-2 satellite DNA in D. gouveai, according Kimura 2 parameters model (Kimura, 1980) Population Ibotirama/BA (J79L67) Pireno polis/go (J18E2) Analândia/SP (J82F3 and J83F2) Altinópolis/SP (H74M2) Peixoto/MG (H27S1) Furnas/MG (H24S3) Total average Average genetic distance Figure 4. Neighbor-joining dendrogram showing the relationships of genetic distance in the repetition units of the pbum-2 satellite DNA analyzed in this work. Only the values of bootstraps above 50% are shown (based on 1000 replicates). The scale bar represents genetic distances calculated according to Kimura 2 parameter method. J79L67 (Ibotirama/BA, locality 1), J18E2 (Pireno polis/go, locality 2), J82F3 and J83F2 (Analândia/SP, locality 6), H74E2 (Altinópolis/SP, locality 5), H24S1 (Furnas/MG, locality 4), H27S1 (Peixoto/MG, locality 3). among five D. seriema populations in Brazil (Kuhn & Sene, 2004). In both cases, no single population, or groups of geographically close populations, could be distinguished by pbum sequences. Such a lack of interpopulational differentiation was discussed in terms of past gene flow events among populations, which is compatible with the results obtained from other genetic markers analyzed in the same populations. Interpopulational analysis of satellite DNA in other insect species also showed high levels of homogenization of the sequences (Bachmann, Venanzetti & Sbordoni, 1994; Bachmann et al., 1998; Lorite et al., 2002). For comparative proposes, the pbum satellite DNA sequences from D. buzzatii (Kuhn et al., 2003) and D. seriema (Kuhn & Sene, 2004) were also analyzed by AMOVA. The results were similar to that found in D. gouveai, i.e., small and insignificant Fct values (Table 2), showing that satellite pbum sequences are extremely conserved on intraspecific level.

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