| چکیده انگلیسی مقاله |
Genus Euphlyctis includes two subgenera, Euphlyctis and Phrynoderma, and more than ten lineages in Southeast Asia. In recent studies, skittering frogs of the genus Euphlyctis have been taxonomically revised. Moreover, some new cryptic species have been identified and described through molecular and morphological approaches. Based on the latest taxonomic revision, related species have been placed into two subgenera, and the status of E. cyanophlyctis (Schneider, 1799) has also been revised. In this study, we aim to elucidate the taxonomic status of the only member of the genus Euphlyctis in Iran, distributed in the southeast of Iran. Additionally, the phylogenetic relationship and genetic divergence of Iran's skittering frogs have also been re-evaluated and discussed. In this study, 32 sequences of mitochondrial 16S rRNA, including 492 bp from Iranian skittering frogs, and 284 sequences from GenBank have been analyzed to clarify the phylogenetic relationships and genetic divergence among populations of the species in Iran and other Asian countries. Phylogenetic analyses using BEAST software revealed 13 clades and subclades within the species. Skittering frogs from Iran were placed in the clade that includes specimens from Pakistan, Nepal, Bangladesh, India, and a topotype of E. adolfi, receiving high support (PP 1.00). In addition, this clade was divided into 3 separate lineages, each of which is related to geographical isolations. Despite molecular analyses, morphological features of skittering frogs from southeast Iran demonstrated variations in morphological characters, resulting in complexity and misleading nomenclature.
Keywords: Skittering frog, Euphlyctis adolfi, Phylogeny, 16S gene, Southern Iran.
Introduction
Genus Euphlyctis belongs to the family Dicroglossidae which includes two subgenera Euphlyctis and Phrynoderma and more than ten lineages in the Southeast Asia (Dubois et al., 2021; Dufresnes et al., 2022; Forest, 2024). Subgenus Euphlyctis consists of E. ehrenbergii, E. jaladhara, E. adolfi, and E. cyanophlyctis distributed over south Asia and E. karaavali, E. hexadactyla, E. aloysii, and E. kerala were included in subgenus Phrynoderma living in the south India, Bangladesh, and Sri Lanka (Dinesh et al., 2021, 2022; Dufresnes et al., 2022). The skittering frogs of Iran can be found in the southeast (Sistan and Baluchestan, and Hormozgan) and were first recognized as Rana cyanophlyctis Boulenger, 1920. Baluch and Kami (1994) mentioned them as R. cyanophlyctis seistanica. Later, it has been revised as E. adolfi (Dufresnes et al., 2022); however, Dinesh et al., (2021) recognized E. adolfi (cyanophlyctis) seistanica as a separate species. In this study, we investigated the phylogenetic relationships and genetic divergence among populations of the species of the genus Euphlyctis in the Southeast Asia.
Materials and methods
A total of 45 individuals of skittering frog (E. cyanophlyctis) were collected from nine localities of Sistan and Baluchestan Province, southeast Iran. Of these, 32 tissue samples from 45 specimens were used for molecular study. Total DNA was extracted from muscle tissue using the phenol/chloroform (Sambrook et al., 1989) and mitochondrial 16S rRNA was amplified using 16SL and 16SH primers (Palumbi, 1991). The final data matrix consisted of 316 Euphlyctis sequences (including 32 sequences from this study and 284 sequences retrieved from GenBank), Fejervarya cancrivora (AB070738) and two Hoplobatrachus tigerinus (AB272594) were used as outgroups. Phylogenetic analysis was conducted using the Bayesian method implemented in BEAST v2.2.1 (Bouckaert et al., 2014) with the GTR+I+G model. All analyses were run in the CIPRES Science Gateway (Miller et al., 2010). The genetic divergences between and within lineages were computed using the p-distance method in Mega.
Research Findings
The Bayesian tree based on 316 Euphlyctis sequences revealed 13 clades and subclades with a posterior probability (PP) of ≥0.70 in South Asia. Clade A includes specimens from Iran, Pakistan, India, Bangladesh, and Nepal. This clade was separated into three subclades (A1, A2, and A3) with high PP (1.00). The specimens from Iran and Pakistan were included in subclade A1. The genetic distances based on p-distance ranged from 1.4% to 1.6% between lineages and 0.2% to 0.3% within subclades. In addition, morphological comparisons of the specimens showed high color polymorphism in the southeast of Iran.
Discussion of Results and Conclusions
Iran's skittering frogs form a clade with specimens from Pakistan, India, Nepal, Bangladesh, and the topotypes of E. adolfi (Clade A), indicating that this clade should be assigned to E. adolfi based on the priority rule. Subclade A1 includes specimens from South Iran and South and Central Pakistan, where two subspecies, E. a. seistanica (Nikolskii, 1899) from Hamoon Saburi in North Sistan, Iran, and E. a. microspinulata Khan, 1997 from Khuzdar, Pakistan, have been described. Considering the close proximity of the sampling site of this study (Karvandr Khash) to Hamoon Saburi (about 200 to 300 km), it can be deduced that Iran's skittering frogs are related to the geographical isolate E. a. seistanica, and E. a. microspinulata is its junior synonym, as supported by morphometric and molecular characters. However, molecular and morphological investigation on the type and paratype specimens is needed to elucidate the taxonomic status of Iran's skittering frogs.
Acknowledgement
This research was supported by grants from the research department of the University of Sistan & Baluchestan, Zahedan, Iran, under project No. 89013. |