Eumeces Wiegmann, 1834 اساس بر ایران، در ) سینسیده : خزندگان ( جنس
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Nova Biologica Reperta 3 (2): 123-130 (2016) 123/123 فیلوژنی مولکولی جنس Eumeces Wiegmann, 1834 )خزندگان: سینسیده( در ایران، بر اساس 16S DNA میتوکندریایی ژن 3 1 *2 1 هیوا فیضی ، نسترن حیدری ، نصراهلل رستگار پویانی و اسکندر رستگار پویانی دریافت: 40/42/1331 / پذیرش: 1331/40/24 1 گروه زیست شناسی، دانشکده علوم، دانشگاه رازی، کرمانشاه، ایران 2 گروه علوم جانوری، دانشکده علوم زیستی، دانشگاه خوارزمی، تهران، ایران 3 گروه زیست شناسی، دانشکده علوم، دانشگاه حکیم سبزواری، سبزوار، ایران * مسئول مکاتبات: [email protected] چکیده. روابط فیلوژنتیکی بین زیرگونه های Eumeces schneiderii princeps و Eumeces schneiderii pavimentatus با استفاده از 143 جفت باز از توالی ژن 16S میتوکندریایی مورد بررسی قرار گرفت. آنالیزها با استفاده از رویکرد حداکثر احتمال (ML) به کمک نرم افزار RAxML بر روی 12 نمونه جمع آوری شده از حدود 24 منطقه مجزا صورت گرفت. نتایج مطالعات مولکولی ما چهار کﻻد کامﻻ مجزا و به خوبی حمایت شده ای از لحاظ موقعیت فیلوژنتیکی، تفاوت ژنتیکی و ویژگیهای مشخص در مورفولوژی و ویژگیهای زیستگاهی آنها تشخیص داد. این کﻻدها شامل گروههایی از (Scincus (3) Eurylepis (2) Eumeces schneiderii princeps+Eumeces schneideri pavimentatus (1 و (Scincopus (4 هستند. همچنین، رابطه فیلوژنتیکی تاکسون ناشناخته .Eumeces sp با کﻻد Scincus بطور کامل مشخص و آشکار نیست. آنالیزهای فیلوژنتیکی صورت گرفته جنس Eurylepis را در کنار کﻻدهای حاوی جنس Eumeces قرار نداد. به عﻻوه، نتایج این آنالیزهای فیلوژنتیکی یک وضعیت مونوفایلتیک را برای گونه Eumeces schneiderii نشان داد. واژههای کلیدی. ایران، بیوجغرافیایی، ژن میتوکندریایی، سینسیده، فیلوژنی، یومسس Molecular phylogeny of the genus Eumeces Wiegmann, 1834 (Reptilia: Scincidae) in Iran, inferred from 16s mitochondrial DNA Hiva Faizi1, Nastaran Heidari2*, Nasrullah Rastegar-Pouyani1 & Eskandar Rastegar-Pouyani3 Received: 23.04.2016 / Accepted: 10.09.2016 1Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran 2Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran 3Department of Biology, Hakim Sabzevari University, Sabzevar, Iran *Correspondent author: [email protected] Abstract. Phylogenetic relationships among the Eumeces schneiderii princeps and Eumeces schneiderii pavimentatus investigated using 509 bp partial sequences of 16S mitochondrial gene. Analyses were done by maximum-likelihood (RAxML) criteria on 52 specimens from over 20 geographically distinct localities. Our molecular results proposed two well-supported major clades by their phylogenetic positions, genetic differences and unique characterizations in their morphology and habitats including: (1) Eumeces schneiderii princeps+ Eumeces schneiderii pavimentatus (2) Eurylepis (3) Scincus and (4) Scincopus. However, the phylogenetic affinities of Eumeces sp. in the Scincus clade were not resolved. Phylogenetic analyses of the genus did not grouped Eurylepis with Eumeces and clustered it in a completely separate group. In addition, phylogenetic results revealed a monophyletic status for Eumeces schneiderii. Keywords. Acanthodactylus, biogeography, cytochrome b, Iran, Lacertidae, mitochondrial genes, ND4, phylogeny Faizi et al. Molecular phylogeny of Eumeces 124/120 INTRODUCTION The name Eumeces schneiderii, initially propos- (Anatolia); E.s. pavimentatus (Geoffroy St. Hilai- ed by Wiegmann in honor of Johann Gottlob Sch- re, 1827) in Syria, Lebanon, Jordan; E.s. prince- neider (1750-1822), German zoologist in Herpeto- ps (Eichwald, 1839) in Armenia, Azarbayejan, logia Mexicana (1834) in that study, three species Caucasus, Iran; Eumeces schneiderii Zarudnyi E. pavimentatus, E. rufescens, and E. punctatus (Nikolsky, 1900) in SE Iranian Plateau (Kerman, recorded under the name of the genus for the first Sistan and Baluchistan provinces), Helmand Bas- time. In a recent study by Griffith et al., (2000), in and southern desert districts of Afganistan, Bal- the genus Eumeces divided into four groups based uchistan and Mekran Coast of Pakistan. In this st- on a series of morphological characteristics analy- udy, the intraspecific phyloge -netic relationships sis and radical changes were proposed for the gen- of Iranian subspecies Eumeces schneiderii prin- us. The paraphyletic genus Eumeces divided into ceps are extracted and will compare with another four separate genera: subspecies extracted from genbank dataset. Eurylepis (“E. taeniolatus” group), Mesoscincus (“E. schwartzei” group), Novoeumeces (“E. schne- MATERIAL AND METHODS ideri” group that includes E. pavimentatus as the Sampling localities for the 52 used specimens in type species of Eumeces sensu lato) and Eumeces this study presented in Figure 1 and Table 1. Eum- (sensu stricto) includes all the other remaining sp- eces schneiderii princeps in its natural habitat sh- ecies, and mainly distributed in East Asia and No- owed in Figure 2 We used Eurylepis, Scincus and rth America. Scincopus as close and distant relatives of the ge- Placement of the genus Eumeces for the species nus as outgroup comparisons to constructing phyl- of North America has emphasized in Griffith wor- ogenetic trees and the relevant sequences down- ks and Lacerta fasciata Linnaeus 1758 chosen as loaded from gene-bank data center (Table 1). type species of the genus Eumeces. In addition, Original DNA and tissue samples deposited in based on cranial traits, Griffith and his colleagues the Department of Biology, Hakim Sabzevari Uni- (2000) recognized Pariocela species group as me- versity. All specimens are deposited in Razi Univ- mbers of the genus Eumeces as the most basic gr- ersity Zoological Museum (RUZM) collection in oup of all skinks throughout the world. In additi- 95% alcohol. on, a new subfamily Eumecinae proposed for this In order to laboratory protocols (DNA extracti- group of species. The proposed new generic na- on, PCR and Sequencing), DNA was extracted fr- me, Pariocela Fitzinger, 1843, for North Americ- om preserved tissue samples using non-organic an skinks of Eumeces (s. l.), later replaced with DNA Extraction Procedure (Proteinase K and Sal- the older available generic name Plestiodon Dum- ting out). 16S gene was amplified with polymera- éril & Bibron, 1839 (Brandley et al., 2005; Schm- se chain reaction (PCR) procedure using 16SL 5´- itz et al., 2004). CGCCTGTTTATCAAAAACAT-3', and 16SH 5'- Moreover, the name Eumeces (sensu stricto) ret- CCGGTCTGAACTCAG ATCACG-3', as primers ained for the group surrounding the type species for 16s mitochondrial gene. that is part of the African-Central Asian clade and PCR reactions were performed in 30µl with the there are currently only five recognized species in following conditions: Initial denaturation stage of this clade has left. 95o C (04:00) followed by the 35 cycles with den- These include Eumeces algeriensis Peters, 1864; aturation at 95oC (00:40), annealing at 49oC Eumeces blythianus (Anderson, 1871); Eumeces (00:40) and extension at 72o C (01:20) then single cholistanensis Masroor, 2009; Eumeces indothale- extension cycle at 72o C (10:00). nsis Khan and Khan, 1997; Eumeces schneiderii The amplified genes were then sequenced with (Daudin, 1802). an automatic DNA sequencer in BIONEER Com- The other (old Eumeces) are now classify in Eu- pany, South Korea following the manufacture pr- rylepis (two species), Mesoscincus (three species, ocedure and protocols. Construction of multiple in North America) and Plestiodon (47 species, in alignments done by Clustal W (Thompson, 1994) North America). So far, five subspecies is known program as implemented in Bioedit software prog- for Eumeces schneiderii (Daudin, 1802) includes ram version 7.0.0 (Hall, 1999); all sequences adju- E. s. barani (Kumlutas et al., 2007) in Turkey sted their ends manually. Nova Biologica Reperta 3 (2): 123-130 (2016) 125/121 Fig. 1. Sampling localities of Eumeces schneideri princeps in this study. Fig. 2. Eumeces schneiderii princeps and its natural habitats in Kordestan and Zanjan provinces, Iran. Assessing substitution saturation done by DAM- Support for the estimated tree assessed using BE software (Xia & Lemey, 2009). The genetic 1000 bootstrap replicates. Bayesian analyses usi- divergences of nucleotide sites among the species ng MrBayes v.3.1.2 used for inferring phyloge- as p-distance in a matrix of pairwise sequences netic relationships among studied taxa and to and the percentage of variable sites and parsimony compare the resulted tree with ML tree. J Model informative sites calculated using MEGA5 (Kum- Test 2.1.1 (Posada, 2008) was used for inferring ar et al., 2008). Maximum likelihood phylogenetic best fitting evolutionary model required for BI an- analysis conducted using RAxMLGUI1.5b1 alyses. The resulted trees visualized using MrEnt- (Silvestro & Michalak, 2012). V.2.4 (Zuccon & Zuccon, 2012). Faizi et al. Molecular phylogeny of Eumeces 126/120 Table 1. Specimens used in this study, collection numbers, GenBank accession numbers, collecting localities and their exact coordination. Collection GenBank Species Coordinates Locality number accession Scincopus fasciatus SCP2 AY64917numbers - - 30km NW Rosso, Mauritania Scincopus fasciatus SCP3 AY30830 - - 30km NW Rosso, Mauritania Scincus scincus SC3 AY712943 - - Unknown 2 Scincus scincus SC4 AY21799 - - Unknown 2 E.s.pavimentatus ES10 JF931189 - - Al Jaboul lake, Syria 6 E.s.pavimentatus ES11 JF931190 - - Azraq, Jordan E.s.pavimentatus ES13 JF931191 - - Um Quays, Jordan E.s.pavimentatus ES14 JF931192 - - Dana Nature Reserve, Jordan E.s.pavimentatus ES17 JF931193 - - Lattakia Beach, Syria E.s.pavimentatus ES18 JF931194 - - Petra, Jordan Eurylepis taeniolatus - JQ344224 - - Unknown Eurylepis taeniolatus