Phylogenetic analysis of the Critically Endangered Karpathos water frog (Anura, Amphibia): Conservation insights from complete mitochondrial genome sequencing

The Karpathos water frog (Pelophylax cerigensis) is classified as Critically Endangered according to the IUCN Red List and its distribution is restricted on the island of Karpathos, South Aegean Sea. Utilizing a next generation sequencing approach, we obtained whole mitochondrial genomes of P. cerigensis to determine the species phylogenetic position within the genus Pelophylax and to clarify the phylogenetic relationship between the species’ population on Karpathos Island and the population of P. cf. bedriagae from the neighboring Rhodes Island. High-throughput sequencing generated mean ± SD = 227,945 ± 18,306 reads averaged per sample. High quality reads were assembled resulting in the complete mitogenome of P. cerigensis of a total size of 17,922 bp. Mitogenome organization was similar to other Pelophylax species, comprising of 13 Protein Coding Genes (PCGs), 22 tRNAs, two rRNAs and one Control Region. Overall mean genetic distance for the 13 PCGs, within the Ranidae family, ranged from 8.01% (COIII) to 11.6% (ATP8), while ratios of synonymous and non-synonymous substitutions were < 1 in all the 13 PCGs, indicating purifying selection. Maximum Likelihood and Bayesian Inference analyses based on the concatenated sequences of 13 PCGs and two rRNAs (12S and 16S), showed that individuals from Karpathos and Rhodes form a well-supported monophyletic group with low genetic distance between them (p-distance = 0.1%). Hence, the Rhodes populations seem to belong to P. cerigensis and not to P. bedriagae as formerly thought, highlighting the need for revision of P. cerigensis current conservation status as well as further examination of the P. bedriagae species group.