**2.1 Species under study**

*Aechmea fulgens* (Figure 2) is a bromeliad native to the Atlantic Rainforest. In addition to its ecological importance, it has economic importance as a plant with ornamental value. The specie was used in this study because of its wide distribution in the Atlantic Rainforest remnants in Pernambuco state and because of its ornamental potential. As certification that the species under study was indeed *Aechmea fulgens*, a specimen from each collection location was identified and placed in the herbarium at the Instituto Agronômico de Pernambuco (IPA 57000.250).

The species and the collection locations were selected following a survey carried out in the herbariums at the Instituto Agronômico de Pernambuco (IPA), the Pernambuco Federal University (UFPE) and the Pernambuco Federal Rural University (UFRPE). The survey considered: species common to the various regions, most recent data collection and importance of the species. The plant material was collected in remnants of the Atlantic Rainforest in three areas located in Pernambuco - region of Northeastern Brazil (Figure 3). Pieces of young leaves were collected from 20 individuals of each population.

#### **2.2 Study areas**

**2.2.1 Charles Darwin Ecological Refuge** (07º49' S - 34º56' W) – located in the region of Igarassu city on the northern coast of Pernambuco state (Brazil), with an area of approximately 60 hectares, whose vegetation is in the process of recomposition.

**2.2.2 Tapacurá Ecological Station** (08º04' S - 35º13' W) - located in eastern Pernambuco state in the region of São Lourenço da Mata city, with about 700 hectares. The mission of the Tapacurá Ecological Station is to carry out research in the fields of botany, zoology and ecology. Work at the station is aimed at developing habitats for conserving forest resources and fauna in the Brazilian Atlantic Forest.

**2.2.3 Mata do Estado** (07o 35'S - 35 º 30'W) - located in the region of São Vicente Férrer city, in north Pernambuco state, with area of approximately 600 hectares . Of the areas studied, this has suffered most from anthropological action.

### **2.3 Isolation of genomic DNA**

Equal amounts (0.2 g dry weight) of leaf tissue from each sample were placed in porcelain mortars refrigerated with liquid nitrogen and ground to a fine powder. Total genomic DNA was extracted according to the CTAB method described by Doyle and Doyle (1990). RNA was removed by RNaseA treatment. DNA samples were spectrophotometrically quantified at 260 nm. The A260/280 reading to each DNA ranged from OD 1.8 to 2.0. After quantification, the DNA was diluted with ultra-pure water to a working concentration of 20 nanograms (ng) µL-1 for PCR analysis.

#### **2.4 SSR assays**

First 12 pairs of primers were selected, developed by Sarthou et al. (2003) and Boneh et al. (2003) for species of the *Pitcairnia, Tillandsia* and *Guzmania* genera, to test the clarity and

*Aechmea fulgens* (Figure 2) is a bromeliad native to the Atlantic Rainforest. In addition to its ecological importance, it has economic importance as a plant with ornamental value. The specie was used in this study because of its wide distribution in the Atlantic Rainforest remnants in Pernambuco state and because of its ornamental potential. As certification that the species under study was indeed *Aechmea fulgens*, a specimen from each collection location was identified and placed in the herbarium at the Instituto Agronômico de

The species and the collection locations were selected following a survey carried out in the herbariums at the Instituto Agronômico de Pernambuco (IPA), the Pernambuco Federal University (UFPE) and the Pernambuco Federal Rural University (UFRPE). The survey considered: species common to the various regions, most recent data collection and importance of the species. The plant material was collected in remnants of the Atlantic Rainforest in three areas located in Pernambuco - region of Northeastern Brazil (Figure 3).

**2.2.1 Charles Darwin Ecological Refuge** (07º49' S - 34º56' W) – located in the region of Igarassu city on the northern coast of Pernambuco state (Brazil), with an area of

**2.2.2 Tapacurá Ecological Station** (08º04' S - 35º13' W) - located in eastern Pernambuco state in the region of São Lourenço da Mata city, with about 700 hectares. The mission of the Tapacurá Ecological Station is to carry out research in the fields of botany, zoology and ecology. Work at the station is aimed at developing habitats for conserving forest resources

**2.2.3 Mata do Estado** (07o 35'S - 35 º 30'W) - located in the region of São Vicente Férrer city, in north Pernambuco state, with area of approximately 600 hectares . Of the areas studied,

Equal amounts (0.2 g dry weight) of leaf tissue from each sample were placed in porcelain mortars refrigerated with liquid nitrogen and ground to a fine powder. Total genomic DNA was extracted according to the CTAB method described by Doyle and Doyle (1990). RNA was removed by RNaseA treatment. DNA samples were spectrophotometrically quantified at 260 nm. The A260/280 reading to each DNA ranged from OD 1.8 to 2.0. After quantification, the DNA was diluted with ultra-pure water to a working concentration of 20

First 12 pairs of primers were selected, developed by Sarthou et al. (2003) and Boneh et al. (2003) for species of the *Pitcairnia, Tillandsia* and *Guzmania* genera, to test the clarity and

Pieces of young leaves were collected from 20 individuals of each population.

approximately 60 hectares, whose vegetation is in the process of recomposition.

**2. Material and methods 2.1 Species under study** 

Pernambuco (IPA 57000.250).

and fauna in the Brazilian Atlantic Forest.

**2.3 Isolation of genomic DNA** 

nanograms (ng) µL-1 for PCR analysis.

**2.4 SSR assays** 

this has suffered most from anthropological action.

**2.2 Study areas** 

Fig. 2. *Aechmea fulgens*. Population of bromeliads in inner of an Atlantic Forest remnant in Igarassu - Pernambuco – Brazil (on the right) and inflorescence (on the left)

Fig. 3. (**a**) Pernambuco State – Brazil; (**b**) Sampling areas for *Aechmea fulgens* populations: ▲Igarassu, north coast; São Lourenço da Mata and ■SãoVicente Férrer, area of forest north.

reproducibility for the analyses. Five pairs of primers were chosen for the amplification reactions that were performed in a TGradient 4.20 Whatman – Biometra (Table 1). With programmed cycles of 5 min at 94°C, followed by 30 cycles of 1 min at 94°C (denaturation); 1 min at 52°C (annealing); and 1 min at 72°C (extension), followed by 7 min at 72oC (final extension of the fragments). Each 20 µL reaction contained 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 100 µM of each triphosphate deoxynucleoside (DNTPs), 0.2 µM of each pair of primers (right and left) from Invitrogen, one unit of the *Taq* DNA polymerase enzyme and 20 ng DNA. The amplification products were resolved in 2.5% agarose gel stained with SyBr Gold (Molecular Probe).
