engleski

Molecular phylogenetic analysis within the genus Rana (Pelophylax) based on three gene markers and RFLP method

Water frogs (genus Rana, subgenus Pelophylax) present an interesting research subject for ecology, physiology and genetics. Rana kl. esculenta hybridogenetic complex, which consists of two parental species (Rana ridibunda and Rana lessonae) and their hybrid species (R. kl. esculenta), is of special interest. Hybrids often show morphological traits similar to either parent, making them harder to identify on the field. Because of that, more relevance is given to molecular and phylogenetic identification methods. In central Croatia both R. ridibunda and R. lessonae parental populations can inhabit the same location and thus lead to the formation of hybrids. The goal of this project was to determine the phylogenetic relationships of water frog populations from Crna Mlaka based on 16S rRNA, cytochrome b and ITS2 sequence analysis, and to examine the efficiency of a simple RFLP method for identifying parental species and hybrids based on the digestion of a conserved ITS2 fragment with three restriction enzymes (KpnI, HaeII and SmaI). Results of this phylogenetic analysis have shown that both parental species R. ridibunda and R. lessonae, and thus potential hybrids, inhabit Crna Mlaka. Phylogenetic analysis of mitochondrial 16S rRNA and cytb genes proved to be the best method for species and haplotype identification, while the RFLP method and phylogenetic analysis of nuclear ITS2 regions, which could help differentiate hybrid samples, need further protocol optimization.

Pelophylax; mtDNA; ITS2; RFLP

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