**2.5 ISSR assays**

The ISSR primers used (Table 2) were selected from a set produced by the University of British Columbia, Vancouver, Canada for *Sphagnum angermanicum* and *Pogonatum dentatum*. The DNA was amplified in a TGradient 4.20 Whatman – Biometra. With the final volume of 25 µL each reaction contained 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 100 µM of each triphosphate deoxynucleoside (DNTPs), 0.2 µM primer from Operon Technologies, one unit of the *Taq* DNA polymerase enzyme and 20 ng DNA. The amplification program was: 94°C for 5 min, 94°C for 30s followed by 30-35 cycles at 50-55 °C, (depending on the primer used). The amplified DNA fragments were separated by electrophoresis in 2.0% agarose gel and stained with SyBr Gold (Molecular Probe).

#### **2.6 Data analysis**

#### **2.6.1 ISSR and SSR markers**

The ISSR marker fragments visualized in the gels were computed as presence (1) or absence (0). The results of these data generated a binary matrix that was analyzed by the POPGENE Program v. 1.31 (Yeh et al. 1999) to estimate some populational parameters: the percentage of polymorphic loci (*P*); *F* statistics by Wright (Φst); Nei genetic diversity (*h*) and the Shannon index (*I*). The Fst values; Φst were used to estimate the gene flow among the populations Nm = 1/4 (1/Fst – 1). The distance between pairs of populations was measured by the Nei genetic distance (Nei 1978). These measurements were then used to construct the dendrogram, using the UPGMA grouping method (Unweighted Pair Group Method with Arithmetic Average). The genetic distance matrix was compared to the geographic distance matrix by the Mantel test to verify whether they were associated. The values of the geographic distance were transformed in log10 for greater proximity to the normal distribution. The Mantel test was carried out by the GENES Program, using 1000 random permutations. The analysis of molecular variance (AMOVA) was used to analyze the distribution of the total within and among genetic variation of the populations, from ISSR marker data using the Program GENES v. 2005.6.1 (Cruz 2001).

The SSR markers were analyzed for the same populational parameters. Because they were co-dominant markers, the data entry, in the genes program, was computed with zero (for absence of fragments), one (for heterozygosity) and 2 (for homozygosity).

#### **3. Results**

The primers used to amplify the microsatellite loci (Table 1) generated, depending on the primer, from one to five fragments per locus with sizes ranging from 110 to 450-base pairs

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

The ISSR primers used (Table 2) were selected from a set produced by the University of British Columbia, Vancouver, Canada for *Sphagnum angermanicum* and *Pogonatum dentatum*. The DNA was amplified in a TGradient 4.20 Whatman – Biometra. With the final volume of 25 µL each reaction contained 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 100 µM of each triphosphate deoxynucleoside (DNTPs), 0.2 µM primer from Operon Technologies, one unit of the *Taq* DNA polymerase enzyme and 20 ng DNA. The amplification program was: 94°C for 5 min, 94°C for 30s followed by 30-35 cycles at 50-55 °C, (depending on the primer used). The amplified DNA fragments were separated by electrophoresis in 2.0% agarose gel

The ISSR marker fragments visualized in the gels were computed as presence (1) or absence (0). The results of these data generated a binary matrix that was analyzed by the POPGENE Program v. 1.31 (Yeh et al. 1999) to estimate some populational parameters: the percentage of polymorphic loci (*P*); *F* statistics by Wright (Φst); Nei genetic diversity (*h*) and the Shannon index (*I*). The Fst values; Φst were used to estimate the gene flow among the populations Nm = 1/4 (1/Fst – 1). The distance between pairs of populations was measured by the Nei genetic distance (Nei 1978). These measurements were then used to construct the dendrogram, using the UPGMA grouping method (Unweighted Pair Group Method with Arithmetic Average). The genetic distance matrix was compared to the geographic distance matrix by the Mantel test to verify whether they were associated. The values of the geographic distance were transformed in log10 for greater proximity to the normal distribution. The Mantel test was carried out by the GENES Program, using 1000 random permutations. The analysis of molecular variance (AMOVA) was used to analyze the distribution of the total within and among genetic variation of the populations, from ISSR

The SSR markers were analyzed for the same populational parameters. Because they were co-dominant markers, the data entry, in the genes program, was computed with zero (for

The primers used to amplify the microsatellite loci (Table 1) generated, depending on the primer, from one to five fragments per locus with sizes ranging from 110 to 450-base pairs

Gold (Molecular Probe).

and stained with SyBr Gold (Molecular Probe).

marker data using the Program GENES v. 2005.6.1 (Cruz 2001).

absence of fragments), one (for heterozygosity) and 2 (for homozygosity).

**2.5 ISSR assays** 

**2.6 Data analysis** 

**3. Results** 

**2.6.1 ISSR and SSR markers** 

(bp). The transferability of the molecular markers was validated among the *Guzmania* and *Pitcairnia* and *Aechmea* genera by amplifying loci of the same size in species of the referred genera of bromeliad. The percentages of polymorphic loci raged, respectively, from 80% to 90.3% for the SSR and ISSR markers and the number of alleles per loci ranged from 1.6 to 1.8 for the three populations (Igarassu, São Lourenço da Mata and São Vicente Férrer) and for the two molecular markers used. Figures 3 and 4 represent an example of the polymorphism obtained with SSR and ISSR primers, respectively.


Table 1. SSR primers, derived species, respective base sequences and amplified fragment size in samples of *Aechmea fulgens.*

Table 2 shows the polymorphism and size of the fragments generated by the ISSR markers and reports the ISSR primers that were used and the percentage of polymorphism produced for the set of populations. The number of fragments ranged from eight to 24 and the fragment sizes ranged from 350-2000 bp. The total molecular genetic diversity for each population was observed both by the percentage of polymorphic loci (*P*) and the Shannon-Wiener (*I*) diversity index and the Nei genetic diversity (*h*).

Fig. 3. SSR marker profiles of 10 samples of *A. fulgens* generated by primer PgI in 2.0% agarose gel. M – 50 bp DNA ladder


The AMOVA analysis, based on 60 ISSR polymorphic loci, showed that 9.1% of the genetic variability was among population and 90.9% within population and the Φst value was, therefore, equal to 0.0903. The Fst (0.040) value detected for the data based on the SSR markers also suggested no differentiation among populations. The estimated gene flow was 2.5 for the analyses with ISSR markers and 5.74 for SSR loci.

Table 2. ISSR primers used and their respective base sequences, number of amplified fragments and polymorphism produced in samples of *Aechmea fulgens.*

Fig. 4. ISSR marker profiles of 10 samples of *A. fulgens* generated by primer UCB 842 in 2.0% agarose gel. M - 1 kb Plus DNA ladder.
