Phylogenetic analysis of the critically endangered Karpathos water frog (Anura, Amphibia): conservation insights from complete mitochondrial genome sequencing

Abstract

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.

Piecing the barcoding puzzle of Palearctic water frogs (Pelophylax) sheds light on amphibian biogeography and global invasions

“…As a consequence, a proper overview of the evolution, diversity and distribution of the genus remains genuinely lacking. Mitochondrial DNA (mtDNA) has been preferentially sequenced to identify and map phylogeographic lineages, but mtDNA sequences are scattered across more than a hundred publications that vary in taxonomic scales, geographic scopes, sets of genes analyzed, and further differ in the way these lineages are delimited, labelled and taxonomically treated (e.g., Akın et al., 2010; Bellati et al., 2023; Bülbül et al., 2011; Dufresnes, Di Santo, et al., 2017; Hoffmann et al., 2015; Komaki et al., 2015; Liu et al., 2010; Lymberakis et al., 2007; Min et al., 2008; Nicolas et al., 2015; Papežík et al., 2023; Plötner et al., 2008; Svinin et al., 2021; Toli et al., 2023; Ualiyeva et al., 2022; Wang et al., 2017). Moreover, mitochondrial barcoding may not be reliable due to hybridization, that cause mitochondrial transfer between sympatric and parapatric species (e.g., Doniol‐Valcroze et al., 2021; Komaki et al., 2015; Liu et al., 2010), including the parental species of hybridogenetic hybrids (Plötner et al., 2008).…”

Genetic Diversity of Pelophylax cerigensis-like Populations on the Anatolian Mainland in Türkiye (Amphibia: Anura: Ranidae)

“…Phylogenetic studies based on one or two mitochondrial genes [6,7,15,17) and complete mitogenomes [16] revealed that P. cerigensis occurs on Karpathos Island but is also present in Rhodes Island. Interestingly, Akın et al [7] and Akın Pekşen [17] indicated that all P. cerigensis specific haplotypes from Karpathos, Rhodes and southwestern Anatolia formed a single monophyletic group.…”

“…In conclusion, both mtDNA and nuclear marker [17] showed that Karpathos and Rhodes Islands and mainland populations of P. cerigensis are closely related. Because of their restricted range and moderate genetic diversity of P. cerigensis population in southwestern Anatolia, they should be a considered as a third Management Unit (MUs) in addition to those as previously suggested for Karpathos and Rhodes populations [16]. It is crucial to develop and implement appropriate conservation strategies to assure future survival and conservation of these threatened P. cerigensis populations.…”

“…bedriagae from East Aegean and Türkiye, not with P. bedriagae from Cyprus and Syria [8,14]. Various past studies [6,7,15,16] indicated that both populations from Karpathos and Rhodes islands shared P. cerigensis-specific haplotypes. Pelophylax cerigensis-specific haplotypes were not only found on Karpathos and Rhodes, but also in the coastal parts of southwestern Anatolia [6,7].…”