Acta Herpetologica 9(2): 159-166, 2014 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-14013 New insights into the taxonomy of the skittering frog Euphlyctis cyanophlyctis complex (Schneider, 1799) (Amphibia: Dicroglossidae) based on mitochondrial 16S rRNA gene sequences in southern Asia Asghar Khajeh1,5, Zeinolabedin Mohammadi2,5,*, Fatemeh Ghorbani2, Javad Meshkani3, Eskandar Rastegar Pouyani3, Adam Torkamanzehi4 1 Department of Crop Productions Technology, Faculty of Agriculture and Natural Resource,, University of Sistan & Baluchestan, Zahedan, Iran 2 Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran. * Corresponding author. E-mail: moham- madi.zeinal@gmail.com 3 Department of biology, Faculty of sciences, Hakim Sabzevari University, Sabzevar, Iran 4 Department of biology, Faculty of sciences, University of Sistan & Baluchestan, Zahedan, Iran 5 Both authors contributed equally to this manuscript Submitted on 2014, 2nd February; revised on 2014, 8th August; accepted on 2014, 12thSeptember Editor: Uwe Fritz Abstract. The Skittering frog (Euphlyctis cyanophlyctis) is considered to be a species complex distributed in south- ern and southeastern Asia. Genetic diversity and taxonomic status of populations across their ranges is unclear and existence of several cryptic species is expected. In this study we used sequence variation in the mitochondrial 16S ribosomal RNA gene to elucidate the taxonomic status of Iranian populations of E. cyanophlyctis and compare their genetic diversity and divergence to populations from the Indian subcontinent. Phylogenetic analysis indicated that the populations of E. cyanophlyctis from Iran, Bangladesh-Assam (northeastern India), southern India, and Sri Lanka are partitioned into different clades. At least four different haplogroups were detected which are here proposed to be considered as allopatric cryptic species. The sedimentation of the Helmand River into the Sistan depression during the Neogene and subsequent formation of dry land barriers are proposed to have caused the Iranian populations of skittering frogs to be disconnected from those of the Indian subcontinent, resulting in differentiation of these line- ages. In addition, some populations from southern India and those from Sri Lanka that were previously recognized as E. cyanophlyctis belong to E. mudigere. A preliminary investigation on the genetic diversity of the populations from southeastern Iran highlights the low genetic diversity among these populations. Keywords. Euphlyctis cyanophlyctis, phylogeny, 16S rRNA gene, differentaition. INTRODUCTION The genus Euphlyctis (family Dicroglossidae) con- sists of six species of which Euphlyctis cyanophlyctis (Sch- neider, 1799) is one of the most widespread in southern Asia. This species has been assigned to various genera over time, including Rana (Rana cyanophlyctis Bouleng- er, 1920), Bufo (Bufo cyanophlyctis Latreille, 1801), and Dicroglossus (Dicroglossus cyanophlyctis Deckert, 1938), and it has been revised in its current name by Dubois (1975). The type locality of this species is probably Tran- quebar in southeastern India (Bauer, 1998) and the spe- cies is believed to be distributed throughout southern Asia from southeastern Iran to Vietnam and Sri Lanka (Frost, 2013). The distribution and taxonomy of Iranian popula- tions of E. cyanophlyctis have been debated by several authors. Boulenger (1920) mentioned that Blanford 160 A. Khajeh et alii recorded it from Makran, Persian Baluchestan. Earlier, Nikolskii (1899) described Rana cyanophlyctis seistanica from Sistan, Iran based on its smaller tympanum and longer snout. This subspecies has only been reported from the type locality and along the border of Iran, Paki- stan, Afghanistan (Khan, 2004). Anderson (1963) later recorded Rana cyanophlyctis from Iran. Baluch and Kami (1995) reported Rana cyanophlyctis cyanophlyctis from southern and southeastern Iran and proposed Rana cyan- ophlyctis seistanica to be a synonym. Additionally, Khan (1997) described a new subspecies, Euphlyctis cyanoph- lyctis microspinulata from Khuzdar, Baluchestan, Pakistan and recognized three subspecies in his checklist of the amphibians of Pakistan (Khan, 2004). The previous phylogenetic analysis by Kosuch et al. (2001) based on a fragment of the mitochondrial 16S ribosomal RNA gene suggested a close relationship between E. cyanophlyctis and E. ehrenbergii. A subsequent analysis suggested a sister relationship between E. cyano- phlyctis and E. hexadactylus (Kurabayashi et al., 2005). Alam et al. (2008) studied the genetic divergence of two species in this genus, E. cyanophlyctis and E. hexadacty- lus, and recognized at least two cryptic species among the populations of E. cyanophlyctis from India, Sri Lanka and Bangladesh. In addition, a new species, Euphlyctis mudi- gere, was described recently from southwestern India (Joshy et al., 2009). During the late Paleocene and early Eocene, the Indian plate, which had broken away from Gondwana in the Early Cretaceous, collided into the Eurasian land- mass and formed the Sistan depression along its west- ern part on the Iranian microcontinent. Later, during the Neogene, the Sistan depression was filled as a result of continuous sedimentation of Helmand River (Molnar and Tapponnier, 1975; Klootwijk and Pierce, 1979). The region is today a transition between the Oriental and Palaearctic faunas and contains a mixture of elements of both (Misonne, 1959; Borkin and Litvinchuk, 2012). However, the Helmand Basin serves as a low altitude bar- rier (Macey et al., 1998; Rastegar Pouyani et al., 2010) between Iranian Plateaue and Indian subcontinent. This area in southeastern Iran comprises dry lands irrigated by the Helmand River and includes Hamoon Lake (Whitney, 2006). The region also comprises a part of the Lut Desert, one of the most arid deserts in the world extending to the Dashti Margo (desert of death) in Afghanistan. Low precipitation and a large excursion between day and nighttime temperatures leads to a low diversity of amphibian in this region except for infre- quent aquatic habitats associated with the Helmand River that provide suitable habitats for populations of E. cyano- phlyctis. In this study, we address the taxonomic status of dif- ferent populations of E. cyanophlyctis from Sistan, south- eastern Iran. We analyzed intraspecific variation within different populations of E. cyanophlyctis of southern Asia and also investigated the phylogenetic relationships among populations of five species of the genus Euphlyc- tis in southern Asia to assess the taxonomic status of the species complex. MATERIALS AND METHODS Specimens A total of 41 individuals of E. cyanophlyctis were col- lected using hand nets in 2010 to 2011 from nine localities in Sistan and Baluchestan province in southeastern Iran (Fig. 1). Identification of specimens was based on Baluch and Kami (1995). All specimens are deposited in the Zoological Labora- tory of Faculty of Agriculture and Natural Resource, Depart- ment of Crop Productions Technology,, Saravan, Iran. Tissue samples were available from 32 specimens of the 41 specimens of E. cyanophlyctis as well as two Pseudepidalea viridis (family Bufonidae) from Iran that were used as outgroups (Table 1 and Fig. 1). All tissue samples were taken from the musculature of the hind limbs and preserved in 80% ethanol. Laboratory procedures Total genomic DNA was extracted from ethanol-pre- served muscle using a standard phenol/chloroform protocol (Sambrook et al., 2001). Standard polymerase chain reaction (PCR) amplifications were performed to amplify 492 bp of the of the mitochondrial 16S ribosomal RNA gene fragment (here- after 16S). PCR amplification was performed using the universal forward primer 16SL (5´-CGCCTGTTTATCAAAAACAT-3´) and the reverse primer 16SH (5´-CCGGTCTGAACTCAGAT- CACG-3´) following the protocol of Palumbi (1991). The amplified DNA fragments were sequenced com- mercially using the automated sequencer ABI prism 3700 at Macrogen Inc. (South Korea). Newly obtained sequences were deposited in GenBank (KF992800 to KF992833) (Table 1). In addition to specimens from Iran, 21 additional 16S sequences of the genus Euphlyctis and one of the dicroglossid Hoplobatrachus tigerinus were obtained from GenBank (Table 1). Phylogenetic analyses Nucleotide sequences were aligned using ClustalW (Thompson et al., 1994) as implemented in the Bioedit sequence alignment editor, v.7.0.9 (Hall, 1999). Gaps in the alignment were treated as missing data. Genetic distances were calculated with MEGA v.4.0 (Tamura et al., 2007) with the p-distances model using the complete deletion option (Kimura, 1980). Phy- logenetic reconstructions were carried out under the maximum 161On the taxonomy of the Euphlyctis cyanophlyctis parsimony (MP) and the maximum likelihood (ML) methods in PAUP* v.4.0b10 (Swofford, 2000). Appropriate substitution models were determined using the Akaike information crite- rion, as implemented in Modeltest (Posada and Crandall, 1998). Maximum likelihood analyses were carried out using a heuristic tree search with 10 random addition sequence replicates and tree-bisection-reconnection (TBR) branch swapping. Maxi- mum parsimony analyses were performed using heuristic search- es with TBR branch swapping and random addition sequence with 1000 replicates. Support for internal nodes was estimated via nonparametric bootstrapping (500 and 5000 replicates for ML and MP, respectively) (Felsenstein, 1985) with a single random addition sequence replicate per bootstrap replicate. Bayesian inference (BI) reconstructions were performed using MrBayes v.3.2.2 (Huelsenbeck et al., 2011). Four simulta- neous Markov chain Monte Carlo (MCMC) chains with incre- mental heating temperature 0.2 were run 6000,000 generations and sampled every 100 generations. The burn-in size was deter- mined by checking the convergence of −log likelihood (−InL) values, and the first 10% of the MCMC chain was discarded. The Bayesian posterior probability (PP) was evaluated from the remaining trees. Haplotype and nucleotide diversity indexes for evaluation of DNA polymorphism were estimated using DnaSP v.5 (Librado and Rozas, 2009). The parsimony network of gene- alogical relationships within E. cyanophlyctis was obtained TCS v.1.21 (Clement et al., 2000). RESULTS Phylogenetic analyses were conducted on a final alignment of 492 bp of 16S for 53 individuals of Euphlyc- tis, as well as two P. viridis and one H. tigerinus that served as outgroups. The alignment contained 81 vari- able sites, of which 48 were parsimony informative. Gaps were coded as missing and there were no ambiguous sites. The best-fit model obtained from Modeltest was a general time-reversible substitution model (GTR+I+G) (-lnL=1782.6420), with a proportion of invariable sites I= 0.3651, variable sites G=0.4488, empirical base fre- quencies (A: 0.3035; C: 0.2413; G: 0.2079; T: 0.2473), and Fig. 1. Map showing the sampling localities of the Euphlyctis cyanophlyctis used for this study. 162 A. Khajeh et alii substitution rates (rate[A–C] 8.0429, rate[A–G] 15.1827, rate[A–T] 6.5556, rate[C–G] 0.9254, rate[C–T] 33.0808, rate [G-T]1.0000) estimated from the data set. These parameters were used for ML analysis. The tree topologies are well resolved and identi- cal among different analyses (Fig. 2). Two strongly sup- ported primary clades (A/B and C) are recognized in all analyses. Clade (A) includes E. cyanophlyctis from Iran (A1) and E. cyanophlyctis from Bangladesh-Assam (A2). Clade (B) consists of E. cyanophlyctis from the southern India (B1), E. ehrenbergii (B2) beside E. mudigere and specimens from India and Sri Lanka that were previously identified as E. cyanophlyctis (B3) (Bossuyt and Milinko- vitch, 2000; Alam et al., 2008). The third clade (C) com- prises sequences of E. aloysii and E. hexadactylus (Fig. 2). The sister relationship between the clade (A1) and (A2) was well supported across all optimality criteria (99% parsimony bootstrap value, 70% likelihood boot- strap and 100% Bayesian posterior probability). In addi- tion, our analyses reveal low genetic distances within each clade, including low inter-population genetic diver- gence between Iran and Bangladesh-Assam specimens (1.7%) and high divergence between Bangladesh-Assam and India (4.7%) (Table 2). Specimens of E. cyanophlyctis from Iran and the southern India had the lowest intra- population genetic distance (0.1%) while specimens from Bangladesh-Assam showed the highest intra-population genetic distance (0.5%) (Table 2). In total, 12 unique 16S haplotypes were identified in 49 individual skittering frogs. Low haplotype diversity was found in Iran populations (Hd= 0.353) in compari- son to higher diversity found in the Bangladesh-Assam populations (Hd= 0.857). Additionally, the populations of skittering frogs from Iran demonstrate the lowest nucleo- tide diversity (π = 0.00072) as compared to the popula- tions from all the other regions. Bangladesh-Assam Table 1. Tissue samples and GenBank accession numbers as well as locality information for the Euphlyctis cyanophlyctis specimens used in this study. Number of specimens (n), number of haplotype (H), haplotype diversity (Hd ±SD) and nucleotide diversity (π±SD). Sequenc- es not generated in the framework of this study were obtained from GenBank from following publications: a: Bossuyt and Milinkovitch (2000); b: Kosuch et al. (2001); c: Kurabayashi et al. (2005); d: Alam et al. (2008); e: Joshy et al. (2009); f: Kotaki et al. (2010); g: Hasan et al. (2012). Sequences with accession numbers starting with KF were generated for the present study. Species Collecting site n Locality number H Hd π Accession Number Country Locality E. cyanophlyctis Iran Karvandar 3 1 2 0.353± 0.084 0.00072 ± 0.00017 KF992818-KF992820 Iran Irendeghan 3 2 KF992821-KF992823   Iran Jalgh 3 3 KF992809-KF992811 Iran Apatan 6 4 KF992800-KF992805 Iran Zaboli 3 5 KF992829-KF992831 Iran Ashar 2 6 KF992827 and KF992828 Iran Sarbaz 3 7 KF992824-KF992826 Iran Domaki 3 8 KF992806-KF992808 Iran Tiran 6 9 KF992812-KF992817 India Padil 2 10 2 0.567± 0.129 0.00262± 0.00066 AB272603-AB272604d India Mangalore 1 10 AB488901f India Konaje 1 11 JF832392 India Madikeri 1 11 AB167938c India Cochin 1 13 AY014366b India Assam 1 18 5 0.857± 0.108 0.00484± 0.00075 AB290420d Bangladesh Mymensingh 3 17 AB272602d, AB530495g, AB272601d Bangladesh Cox’s Bazar 4 16 AB530494g, AB530496-AB530498g E. mudigere Sri Lanka 2 15 3 0.833 ± 0.222 0.00378± 0.00378 AB290418-AB290419d India Mudigere 2 12 AB377109e, AF249053a E. aloysii India Adyar 1 14 - - - AB272606e E. ehrenbergii Yemen 1 - - - - AY014367 b E. hexadactylus India Mangalore 1 10 - - - AB167941 c H. tigerinus India Padil 1 10 - - - AB272594 d P. viridis Iran Zabol 2 - - - - KF992832-KF992833 163On the taxonomy of the Euphlyctis cyanophlyctis Fig. 2. Maximum likelihood phylogeny for Euphlyctis cyanophlyctis (using a 492 bp portion of the mitochondrial 16S ribosomal RNA gene). Numbers represent MP and ML bootstrap values (MP/ML), respectively (500/5000 replicates). The posterior probability values from the Bayesian analysis are indicated at the > 99 % (**) and > 95 % (*) significance levels. 164 A. Khajeh et alii populations have the highest nucleotide diversity (π = 0.00484). Iran populations exhibit the lowest number of 16S rRNA haplotypes (H=2) whereas the highest number of 16S rRNA haplotypes was found in Bangladesh-Assam (H=5). These 12 haplotypes were grouped in four sepa- rate haplogroups that correspond to the geographic dis- tribution of these samples (Table 1 and Fig. 3). The first haplogroup includes two haplotypes from Iran and the second haplogroup was identified as related to nominal E. cyanophlyctis from the southern India. The third hap- logroup consists of five 16S rRNA haplotypes from Bang- ladesh-Assam specimens and the fourth haplogroup has three haplotypes that included specimens from Sri Lanka, southern India, and one specimen of E. mudigere (Fig. 3). DISCUSSION The phylogenetic analyses, genetic distances, and haplotype network analyses strongly suggest that E. cyan- ophlyctis populations are split into four main genetic lin- eages separated by a high level of nucleotide divergence (between 1.7% and 4.7% K2P distance). These lineages correspond to the eastern clade (A1) from Iran, the northern clade (A2) specimens Bangladesh-Assam, the southern clade (B1) that we consider to be nominal E. cyanophlyctis because of its proximity to the type local- ity, and the clade from southern India and Sri Lanka that includes E. mudigere. Morphometric, morphological and molecular simi- larities (low haplotype and low nucleotide diversity) between populations of skittering frogs from southeast- ern Iran casts strong doubts on the validity of E. cyano- phlyctis seistanica. In contrast, there is substantial diver- gence between populations of E. cyanophlyctis from Iran and the Indian subcontinent. Formation and subsequent sedimentation of the Sistan depression during the Neo- gene (Whitney, 2006) might have played a role as the main barrier, causing geographic isolation and divergence between populations of skittering frogs on the Indian subcontinent and in the Iranian Helmand Basin. Genetic divergence is a major consequence of vicariance and both dispersal events and isolation by distance can drive spe- ciation (Lomolino et al., 2005). The formation of the Hel- mand barrier was proposed to have driven vicariance of different clades within the genus Laudakia and the Eremi- as persica complex (Macey et al., 1998; Rastegar Pouyani et al., 2010). The close relationship of specimens from Iran and Bangladesh is difficult to explain in terms of geological and dispersal events. This could be the result of molecu- lar homoplasy or ancestral state polymorphism (Avise, 1994) caused by recent fragmentation rather than recent dispersion. On the other hand, the specimens from Sri Lanka and India constitute a monophyletic group with E. mudigere suggesting that all of these specimens are refer- able to that species. The occurrence of E. mudigere in Sri Lanka and India was not unexpected since Sri Lanka is a part of the Deccan Plateau and was connected to the Indian subcontinent until the late Miocene (Deraniyaga- la, 1992). Although specific values of genetic divergence should not serve as the only basis for delimitation of spe- cies, a threshold of 3% genetic divergence could be used for recognition of a new species (based on Fouquet et al. 2007 for 16S). We recovered genetic divergences more than 3% between Bangladesh-Assam and southern Indian (4.7%) as well as between Iran and Indian populations (4.1%). These results are concordant with the study by Alam et al. (2008) that identified several cryptic species including at least four cryptic species currently identified as E. cyanophlyctis. These cryptic lineages are probably the result of allopatric speciation and a detailed taxonom- ic revision is strongly recommended. Table 2. P-distances of the 16S gene of Euphlyctis species. Within- group divergences in bold. Species 1 2 3 4 5 6 7 1. E. cyanophlyctis (Iran) 0.1 2. E. cyanophlyctis (Bangladesh-Assam) 1.7 0.5 3. E. cyanophlyctis ( India ) 4.1 4.7 0.1 4. E. mudigere 3.8 4.5 3.4 0.4 5. E. ehrenbergii 6.7 7.4 5.4 5.1 - 6. E. hexadactylus 9.9 10.3 9.8 9.9 11.5 - 7. E. aloysii 10.8 10.7 11.1 10.2 11.5 7.7 - Fig. 3. Haplotype networks of Euphlyctis cyanophlyctis (49 individu- als) based on a 492 bp portion of the mitochondrial 16S ribosomal RNA gene. Networks were not joined if haplotypes were separat- ed by more than 9 mutations. (h1, h2): Iran; (h3, h4): India; (h5, h6, h7, h8, h9): Bangladesh- Assam; (h10, h11, h12): Mudigere (AB377109), India and Sri Lanka. 165On the taxonomy of the Euphlyctis cyanophlyctis ACKNOWLEDGMENTS This research was supported by grants from the research department of the University of Sistan & Bal- uchestan, Zahedan, Iran under project No. 89013. Per- mission to collect was authorized by the Iran Depart- ment of Environment (Permission Number: 912-N/13/1; 2009; 15th August). We are grateful to Professor Muham- mad Sharif Khan for his kind cooperation in identifica- tion of the specimens. Our special thanks go to David C. 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