Materials and Methods Laboratory Techniques

Blood samples were taken in the field (or from captive specimens of known collection locality) by caudal puncture (see Joger and Lenk 1997). The animals were released after photographic documentation. DNA was also extracted from ethanol preserved tissues. We used a standard method for obtaining total genomic DNA (Sambrook and Russell 2001).

As a gene tree is not necessarily identical to the species tree (see Avise 1994), we analyzed both mitochondrial and nuclear markers, wherever possible. Mitochondrial DNA, predominantly cytochrome b, was amplified by PCR and directly sequenced. (see the cited original publications for primer sequences). The applied nuclear markers were either allozymes and plasma proteins, or the interspaces between microsatellites - ISSRs (see Joger et al. 2006, 2007).

Both kinds of markers supplement each other and their combination thus enhances the capacity to resolve the phylogeographical history. Mitochondrial sequences are powerful tools to reconstructing subsequent multiple splitting events. Moreover, as they are inherited only maternally, without recombination, introgres-sions lead to the coexistence of several matrilines (haplotypes), thus preserving multiple origins of populations. On the other hand, hybridization (e.g., in secondary contact zones) cannot be detected by mitochondrial DNA. Bisexually inherited nuclear markers do not have this restriction.

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