Hydromedusa maximiliani is a vulnerable neotropical freshwater turtle endemic to mountainous regions …

• Abstract
• Introduction
• Materials and Methods
• Results
• Discussion
• References
• Figure and Tables

Field work was conducted from November 1998 to November 1999 at the Parque Estadual de Carlos Botelho (PECB), state of São Paulo, southeastern Brazil (24°00-24°15S, 47°45-48°10W). The PECB is a protected reserve that encompasses over 37,000 ha of intact tropical montane rain forest typical of southeastern Brazil (Whitmore, 1990; Veloso et al., 1991). In this study, an area of approximately 500 ha containing eight rivers and streams (1-8) was surveyed (Figure 1). This 500 ha area was the same as used in previous studies of this species natural history [see Souza and Abe (1995, 1997a,b, 1998) for a more detailed description of the area]. Three sites were defined within these drainage based on the spatial hierarchy of the main rivers and their tributaries: site A (which included river 1), site B (which included rivers 2, 3, 4, and 5), and site C (which included rivers 6, 7, and 8) (Figure 1).

Turtles (n = 25) were hand-caught and 200-300 mL of blood was drawn from the scapula vein/brachial artery (Avery and Vitt, 1984) using a 26-gauge needle and a 1 mL syringe. The blood samples were immediately preserved in 1 mL of absolute ethanol (Miyaki et al., 1997) in plastic vials and stored at room temperature. The turtles were released at the point of capture after blood sample collection.

Genomic DNA was extracted from the blood samples by two successive organic extractions with phenol:chloroform:isoamyl alcohol as outlined by Bruford et al. (1992) and Miyaki et al. (1998), and then precipitated with 1/10 volume of 1 M sodium acetate (pH 5.3) and two volumes of 100% ethanol. The quality of the extracted DNA was evaluated in agarose gels (0.7%) stained with ethidium bromide and quantified by comparison with DNA standards run in the same gel. DNA was diluted to a working concentration of approximately 0.6 ng/mL.

Eighty primers (Operon Technologies, Inc.; Alameda, California, EUA) were initially screened for consistently reproducible and scoreable amplified bands. Nine primers that met these criteria were used to analyze the DNA samples from the 25 individuals captured in the studied area. The polymerase chain reaction (PCR) was performed in a Perkin-Elmer GeneAmp^TM PCR system 9700 with a total volume of 12.5 mL containing 10 mM Tris HCl (pH 8.4), 50 mM KCl, 3.5 mM MgCl₂, 1 mL of each dNTP, 2 mL of primer, 0.3 mL Taq polymerase, 1.2 ng of genomic DNA and sterile water. Negative controls in which water was substituted for DNA were run to check for the possibility of contamination. Reproducibility was gauged by comparing duplicate reactions, which were usually adjacent to one another in the thermocycler, and products were run side-by-side on the same gel. The reaction conditions involved initial denaturation of DNA for 2 min at 94 °C, 39 cycles of 1 min denaturation at 94 °C, 1 min annealing at 40 °C, 2 min extension at 72 °C, and one 5 min cycle at 72 °C for final extension. The amplification products were separated on 1.3% agarose gels stained with ethidium bromide, run in buffer 1X TAE at a constant voltage of 80 V for 5 h. Monochrome photographic negatives were taken of the gels and the individual profiles were scored by two of the authors (FLS and AFC) for the presence/absence of fragments for each primer (see Souza et al., 2002, for a full description of these methods).

Allele frequencies were estimated for standard genetic analysis of population structure. Most (~90%) alleles amplified by arbitrarily primed PCR segregate as dominant markers. Since these RAPD alleles are revealed as the presence or absence of a band, it is not generally possible to distinguish heterozygous individuals from those homozygous for the dominant allele at such loci because both have the "band present" phenotype (Ferreira and Grattapaglia, 1998). We assumed that all loci considered in our analyses met this criterion and that the genotypes were in Hardy-Weinberg equilibrium. Allele frequencies were obtained using the asymptotically unbiased estimator,, derived by Lynch and Milligan (1994), as follows

where is the frequency of the null allele calculated as the square root of the frequency of the null phenotype (i.e., absent band).

The analysis of molecular variance, AMOVA (Excoffier et al., 1992) was used to measure the variation in allelic frequencies for each locus among populations of turtles inhabiting different rivers and streams from the sampled drainage. Gene flow (Nm) was estimated from F-statistics, F_ST, as a measure of the genetic interaction among populations, indicating the number of immigrants per population per generation (Slatkin, 1985, 1987). We used the formula F_ST = 1/(4Nm + 1), where N is the local population size and m is the average rate of immigration. For this estimate, we assumed neutrality, negligible mutation and a stepping-stone population structure model (Kimura and Weiss, 1964). This assumption was crucial because the relation of F_ST to underlying microevolutionary parameters changes with different models of population structure (Slatkin, 1985, 1987). Contingency chi-square values were calculated to determine whether F_ST estimates varied from zero (significant population differentiation), using the formula c² = 2N F_ST(k-1), where N is the total sample size, and k is the number of alleles; df = (k-1)(m-1), where m is the number of samples (in this case, the number of sites within sampled drainage) (Johnson and Black, 1991).