ZooKeys 748: 97-1 29 (20 | 8) A peer-reviewed open-access journal doi: 10.3897/zookeys.748.20507 RESEARCH ARTICLE #ZooKey S http:/ / ZOO keys -pen soft. net Launched to accelerate biodiversity research Molecular and morphological differentiation of Secret Toad-headed agama, Phrynocephalus mystaceus, with the description of a new subspecies from Iran (Reptilia, Agamidae) Evgeniya N. Solovyeva', Evgeniy N. Dunayev', Roman A. Nazarov', Mehdi Radjabizadeh’, Nikolay A. Poyarkov, Jr.? I Zoological Museum of the Lomonosov Moscow State University, Bolshaya Nikitskaya st. 2, Moscow 125009, Russia 2 Department of Biodiversity, Institute of Environmental Science, International Center for Science, High Technology and Environmental Science, Kerman, Iran 3 Department of Vertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Leninskiye Gory, GSP-1, Moscow 119991, Russia Corresponding author: Evgeniya N. Solovyeva (anolis@yandex.ru); Nikolay A. Poyarkov (n.poyarkov@gmail.com) Academic editor: J. Penner | Received 22 August 2017 | Accepted 23 March 2018 | Published 5 April 2018 http://zoobank. org!7933C253-2665-41 91-87 1D-BF653A82FE06 Citation: Solovyeva EN, Dunayev EN, Nazarov RA, Radjabizadeh M, Poyarkov Jr NA (2018) Molecular and morphological differentiation of Secret Toad-headed agama, Phrynocephalus mystaceus, with the description of a new subspecies from Iran (Reptilia, Agamidae). ZooKeys 748: 97-129. https://doi.org/10.3897/zookeys.748.20507 Abstract The morphological and genetic variation of a wide-ranging Secret Toad-headed agama, Phrynocephalus mystaceus that inhabits sand deserts of south-eastern Europe, Middle East, Middle Asia, and western China is reviewed. Based on the morphological differences and high divergence in COI (mtDNA) gene sequences a new subspecies of Ph. mystaceus is described from Khorasan Razavi Province in Iran. Partial sequences of COI mtDNA gene of 31 specimens of Ph. mystaceus from 17 localities from all major parts of species range were analyzed. Genetic distances show a deep divergence between Ph. mystaceus khorasanus ssp. n. from Khorasan Razavi Province and all other populations of Ph. mystaceus. The new subspecies can be distinguished from other populations of P/. mystaceus by a combination of several morphological features. Molecular and morphological analyses do not support the validity of other P/. mystaceus subspe- cies described from Middle Asia and Caspian basin. Geographic variations in the Ph. mystaceus species complex and the status of previously described subspecies were discussed. Keywords Khorasan, molecular phylogenetics, morphology, phylogeography, Phrynocephalus mystaceus khorasanus, taxonomy Copyright Evgeniya N. Solovyeva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 98 Evgeniya N. Solovyeva et al. / ZooKeys 748: 97-129 (2018) Introduction Toad-headed agamas of the genus Phrynocephalus Kaup, 1825, are distributed from south-eastern Europe and southwest Asia (including the Middle East and Arabian Pen- insula) through Middle Asia to Central Asia (northern and central China and Mongo- lia). This taxonomically complicated genus currently contains up to 32 species (Uetz and Hosek 2016). The secret toad-headed agama, or Phrynocephalus mystaceus (Pallas, 1776), is one of the largest representatives of the genus, and is easily distinguished from all other congeners by a pair of large fringed cutaneous folds at the mouth angles. It is a specialized psammophilous species that inhabits sand dunes from Caspian region of the south-eastern part of European Russia in the west to the Ili valley in eastern Kazakhstan and western China in the east, and from Kazakhstan in the north through Middle Asia to northeastern Iran in the south (Bannikov et al. 1977; Zhao and Adler 1993; Anderson 1999; Ananjeva et al. 2004; Molavi et al. 2014; Fig. 1). Phrynocephalus mystaceus was shown to have a high level of anatomical variabil- ity (Ananjeva “1986” 1987), which, together with its unique karyotype (Zeng et al. 1997), has led to its uncertain taxonomic classification at the generic level. Eichwald (1831) proposed the new generic name Megalochilus Eichwald, 1831 for Ph. mysta- ceus, which was synonymized with the genus Saccostoma by Fitzinger (1843). Ananjeva (“1986” 1987) restored the monotypic genus Megalochilus, but such taxonomic change was contradicted by Golubev and Sattorov (1992), as they argued that the differences proposed by Ananjeva were too slight to warrant a separate genus status. Molecular phylogenetic analyses based on mtDNA markers failed to resolve the phylogenetic po- sition of Ph. mystaceus (Pang et al. 2003), which led Barabanov and Ananjeva (2007) to consider Megalochilus as a junior synonym of Phrynocephalus. However, a recent phy- logeny based on the analysis of RAG/ nuDNA gene indicated Ph. mystaceus as a sister lineage with respect to all other examined Phrynocephalus species (Melville et al. 2009). Further study with better taxon sampling based on mtDNA data suggested that P/. mystaceus is a member of the “core” Phrynocephalus clade and is associated with Ph. ax- illaris (Solovyeva et al. 2014). The most recent study proposed to consider Megalochilus as a subgenus of the genus Phrynocephalus (Solovyeva et al. 2014). There was little consensus in the understanding of intraspecific taxonomy of P/. mystaceus. Krassowky (1932) was the first to split P+. mystaceus into two subspecies: European nominative subspecies Ph. m. mystaceus (Pallas, 1776) and Middle-Asian subspecies Ph. m. galli Krassowsky, 1932. This taxonomic classification was supported by subsequent studies of Soviet herpetologists (Shibanov 1941; Terentjev and Chernov 1949; Khonyakina 1961). However, morphometric studies by Vel'dre (1964a, 1964b) suggested that it is impossible to distinguish geographical races within Ph. mystaceus due to its high morphological variability among populations. Consequently, Ananjeva (1987 “1986”) suggested to upgrade the Middle-Asian subspecies Ph. m. galli to full species status and recognized a distinct subspeciesin Daghestan (Megalochilus mystaceus dagestanica in Ananjeva et al. 1987 “1986”). Semenov and Shenbrot (1990) analyzed morphological and chromatic differentiation of Ph. mystaceus from “Semirechye” (an Molecular and morphological differentiation of Secret Toad-headed agama... 99 area east of lake Balkhash in Eastern Kazakhstan), and suggested that this area is inhab- ited by a distinct subspecies, Ph. mystaceus aurantiacocaudatus Semenov et Shenbrot, 1990, which differs from the Middle Asian subspecies Ph. m. galli by its bright orange- red coloration of the ventral surface of the tail in young specimens (versus lemon-yellow coloration in other subspecies). However, Ph. mystaceus aurantiacocaudatus was syn- onymized with P/. m. galli by Barabanov and Ananjeva (2007) without any discussion. In summary, three subspecies of Phrynocephalus mystaceus are recognized in recent literature (see Barabanov and Ananjeva 2007): 1. Ph. m. mystaceus (Pallas, 1776), that inhabits eastern Ciscaucasia (eastern part of Chechen Republic, Daghestan, Kalmykia), Caspian region (southern part of As- trakhan Region, east of the Volga-Ural Sands; introduced to the Apsheron Pen- insula, Azerbaijan) and northwestern Kazakhstan (Ananjeva et al. 2004). Terra typica restricta: Ryn-Peski (Ryn Sands), Ural Region, northwestern Kazakhstan (Barabanov and Ananjeva 2007). This form includes Megalochilus mystaceus dagest- anica Ananjeva, “1986” 1987, described from Kumtorkala, Daghestan, Russia, as a junior synonym. 2. Ph. mystaceus galli Krassowsky, 1932, that inhabits Transcaspian Region and Mid- dle Asia from Turkmenistan, Uzbekistan, Kazakhstan, to northeastern and eastern Iran and adjacent areas of Afghanistan (Anderson 1999; Ananjeva et al. 2004). Terra typica: Repetek station, Lebapsky District, Turkmenistan (Barabanov and Ananjeva 2007). Based on its distribution, this subspecies is supposed to inhabit north-eastern Iran (Anderson 1999). 3. Ph. mystaceus aurantiacocaudatus Semenov & Shenbrot, 1990, known from eastern Kazakhstan and western China (Ili River Valley in Xinjiang). Terra typica: 70 km north northwest of Ushtobe, Eastern Kazakhstan. Regarded as a junior synonym of Pf. mystaceus galli by Barabanov and Ananjeva (2007), however, without any justification. It is notable that all previous works on geographic variations of Ph. mystaceus omit- ted populations from the southernmost edge of its range, Iran and Afghanistan, from the analyses. Morphological characterization and analysis of distribution of Ph. mysta- ceus in Iran was carried out by Anderson (1999) and Molavi et al. (2014). Anderson (1999) examined specimens from Iran and Uzbekistan, and proposed that Iranian populations demonstrate intermediate morphology between Ph. m. galli and Ph. mys- taceus. Molavi et al. (2014), based on a study of seven specimens from Semnan Prov- ince, repeated earlier conclusions by Anderson (1999) and suggested that further in- vestigation of both morphological and molecular characters are required to clarify the taxonomic status of Iranian Ph. mystaceus populations. The recent analysis of phylogenetic relationships within the genus Phrynocephalus based on four mitochondrial genes revealed a remarkable divergence between Ph. mys- taceus samples from Iran and Middle Asia (Solovyeva et al. 2014). Based on these re- sults the Iranian population was tentatively indicated as a putative new subspecies P/. mystaceus ssp. In the present study, we provide a detailed analysis of both morphologi- 100 Evgeniya N. Solovyeva et al. / ZooKeys 748: 97-129 (2018) cal and genetic variation of Ph. mystaceus across its range and confirm deep differentia- tion between the population from Khorasan Province of Iran and other populations in the species range. The currently recognized subspecies of Ph. mystaceus are reviewed and a new subspecies from Khorasan Province is described, based on both molecular and morphological features. Materials and methods Sampling. Historical collections of the Zoological Museum of Lomonosov Moscow State University (ZMMU) were examined, in total, 70 adult and subadult specimens of all currently recognized subspecies (Appendix 1). In addition, type specimens of Ph. mystaceus galli (lectotype, ZMMU R-6413) and Ph. mystaceus aurantiacocaudatus (holotype, ZMMU R-6412) were also examined. Sampling was carried out in the Khorasan Province of Iran in April of 2005, April of 2006, May and June of 2009, and May of 2010. Specimens from Iran were obtained through the collaboration with the Zoological Museum of International Center for Science, High Technology and Envi- ronmental Sciences (ICSTZM; Kerman, Iran; MOU no. 158/2010). Tissue samples from 31 Ph. mystaceus specimens were used in molecular analyses, and their geographic distribution is shown in Fig. 1. Details on museum IDs and localities of origin for each sample are summarized in Table 1. Molecular analyses. Mitochondrial DNA COI gene (cytochrome oxidase ¢ sub- unit I) fragment, 654 b. p. in length was analyzed. Muscle and skin tissues were disintegrated with Proteinase K and total genomic DNA was extracted using a stan- dard phenol-chloroform extraction protocol followed by ethanol precipitation of DNA (Sambrook et al. 1989). PCR amplification was performed using MyCycler BioRad under conditions described by Ivanova et al. (2006). Standard pair of prim- ers was used: VFld (5'-TTCTCAACCAACCACAARGAYATYGG-3') and VR1d (5'-TAGACTTCTGGGTGGCCRAARAAYCA-3') or Rep-COI-F (5'-TNTT- MTCAACNAACCACAAAGA-3') and Rep-COI-R_ (5'-ACTTCTGGRTGKC- CAAARAATCA-3'). PCR reaction volume was 20 ul and it contained ca. 100 ng of template DNA, 0.3 pM/pl of each PCR primer, 1xTaq-buffer with 25 mM of MgCl, (Silex, Moscow Russia), 0.2 mM dNTPs, and 1 unit of Tag-polymerase (Si- lex, Moscow Russia; 5 units/pl). The results of the amplification were examined us- ing electrophoresis in 1% agarose gel in presence of ethidium bromide. The length of the obtained fragments was 680 bp. We included two sequences of Ph. mysta- ceus from western China available from Genbank (NC022131 and KC578685; see Chen et al. 2014) in the analyses. Samples of Ph. melanurus (ZMMU R-12328, Gen- Bank AN MF567976) and Trapelus sanguinolentus (ZMMU R-12709, GenBank AN KF691668) were used as outgroups. Sequences were aligned using Seqman 5.06 and checked using BioEdit Sequence Alignment Editor 7.1.3.0 (Hall 1999). All sequences were deposited in GenBank (see Table 1 for all voucher information, with corresponding GenBank accession numbers). Molecular and morphological differentiation of Secret Toad-headed agama... 101 @ Ph. m. mystaceus OPh. m. “aurantiacocaudatus” @ Ph. m. “galli” @ Ph. m. khorasanus ssp. n. © terra typica Asst Figure |. Geographical distribution of Phrynocephalus mystaceus and locations of the sites where the sam- ples that were examined in the molecular analyses of the present study were obtained. Locality numbers correspond to those given in Table 1. Dot in the center of a circle indicates the type locality; type localities for taxa are shown as follows: A Lacerta mystacea Pallas, 1776 B Megalochilus mystaceus dagestanica Anan- jeva, “1986” 1987 C Phrynocephalus mystaceus aurantiacocaudatus Semenov & Shenbrot, 1990 D Phryno- cephalus mystaceus galli Krassowsky, 1932; and E Ph. mystaceus khorasanus ssp. n. Mean uncorrected p-distances and sequences characteristics were calculated using MEGA 6 (Tamura et al. 2011). Phylogenetic analyses were conducted using Treefinder (Jobb et al. 2011) and MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) software. PartitionFinder v1.0.1 (Lanfear et al. 2012) was used to estimate the optimal evo- lutionary models for Bayesian inference analysis. The preferred model for CO/ align- ment was HKY + G for two partitions (codon position 1 and 2 vs. codon position 3) as suggested by the Akaike information criterion (AIC). Bayesian phylogenetic analysis was performed using MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003) with two simultaneous runs, each with four chains, for 20 million generations, 2 million genera- tions were cut as burn in. The convergence of the runs was checked to make sure that the effective sample sizes (ESS) were all above 200 by examining the likelihood plots using TRACER v.1.5 (Rambaut and Drummond 2007). The Maximum Likelihood (ML) analysis was conducted using Treefinder (Jobb et al. 2011). Each dataset was divided into three partitions according to codon positions; for each partition the best fitting substitution model was selected using the AIC in Treefinder. For ML-analysis we used 1000 pseudoreplics (BS) and Expected Likeli- hood Weights (ELW). 102 Evgeniya N. Solovyeva et al. / ZooKeys 748: 97-129 (2018) Table |. List of the samples used in molecular analyses. Locality numbers correspond to those in Figure 1. Voucher N° Subspecies Locality GenBank N° ZMMU R-12202 Ph. mystaceus khorasanus ssp. n.| Iran, Khorasan Razavi Prov., Gonabad (1) MF567983 ZMMU R-13009-1 | Ph. mystaceus khorasanus ssp. n. Iran, Khorasan Razavi Prov., Boshrue (2) MF567989 ZMMU R-13009-2. | Ph. mystaceus khorasanus ssp. n. Iran, Khorasan Razavi Prov., Boshrue (2) KF691714 ZMMU R-13011-1 | Ph. mystaceus khorasanus ssp.n.| Iran, Khorasan Razavi Prov., Gonabad (1) MF567987 ZMMU R-13011-2. | Ph. mystaceus khorasanus ssp.n.| Iran, Khorasan Razavi Prov., Gonabad (1) MF567988 ZMMU R-11913 Ph. mystaceus khorasanus ssp. n.| Iran, Khorasan Razavi Prov., Gonabad (1) MF567975 ZMMU R-13169 | Ph. mystaceus khorasanus ssp. n. tea eee ae hoon MF567974 Gonabad (3) RuHF-072-1 Ph. mystaceus mystaceus Russia, Astrakhan Prov., Dosang (4) MF567968 RuHE-072-2 Ph. mystaceus mystaceus Russia, Astrakhan Proy., Dosang (4) MF567969 ZMMU-R-12457-2 Ph. mystaceus mystaceus Russia, Astrakhan Proy., Dosang (4) MF567990 ZMMU-R-12457-3 Ph. mystaceus mystaceus Russia, Astrakhan Proy., Dosang (4) MF567986 Kazakhstan, N Priaralye, S border of Malye RuHF-079-1 Ph. mystaceus galli Baekicias() MF567971 ; Kazakhstan, N Priaralye, S border of Malye RuHF-079-2 Ph. mystaceus galli Batsulieands (5) MF567970 ZMMU-R-12517-2 Ph. mystaceus galli Bran Tea yo peraes Oo Mate | Sipsaroen Barsuki sands (5) Dans ZMMU R-12772 Ph. mystaceus galli Kazakhstan, Aralsk (6) MF567982_ ZMMU R-12775 Ph. mystaceus galli Uzbekistan, Qarakalpaqiston Republic (7) | MF567981 s Uzbekistan, Qarakalpaqiston Republic, ZMMU-R-12266 Ph. mystaceus galli Chukurkak (8) MF567978 ZMMU-R-12252-1 Ph. mystaceus galli Uzbekistan, Navoi Prov., Terankuduk (9) MF567977 ZMMU-R-12261-1 Ph. mystaceus galli Rogers aya er Ramaniiha desi KF691713 ZMMU R-12799 Ph. mystaceus galli Tajikistan, Shaartuz (11) MF567979 RuHF-077-1 Ee my Notte E Kazakhstan, N Kapchagai Reservoir (12) | MF567972 aurantiacocaudatus RuHF-077-2 fe ies ent E Kazakhstan, N Kapchagai Reservoir (12) | MF567973 aurantiacocaudatus Ph. mystaceus SE Kazakhstan, left bank of Ili River,125 km i ft ’ >’ ME 4 AMINO R258 aurantiacocaudatus of the road Almaty-Bakanas (13) ae ZMMU R-12778 ikieecgs Kazakhstan, Pidzhim env. (14) MF567980 aurantiacocaudatus ZMMU R-14715-1 ET Sues Kazakhstan, S Balkhash lake, N of Matay (15) | MEF567991 aurantiacocaudatus ZMMU R-14715-2 Ei ceatis) Kazakhstan, $ Balkhash lake, N of Matay (15) | MF567992 aurantiacocaudatus ZMMU R-14715-3 EP TOs Kazakhstan, $ Balkhash lake, N of Matay (15) | MF567993 aurantiacocaudatus ZMMU R-14715-4 He Aes paciine Kazakhstan, S Balkhash lake, N of Matay (15) | MF567994 aurantiacocaudatus 7MMU NAP-05510 Ph. mystaceus Kazakhstan, E Balkhash lake, environs of MF567995 aurantiacocaudatus Kabanbay (16) No voucher number Lin ayia China, Ili River valley, Huocheng (17) NC021131 aurantiacocaudatus Molecular and morphological differentiation of Secret Toad-headed agama... 103 Confidence in tree topology was tested by using non-parametric bootstrap analysis (Felsenstein 1985) with 1000 replicates and posterior probability (PP) for Bayesian inference (BA) in MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001). Branches with bootstrap values of 70% or higher and posterior probabilities values over 0.95 were regarded as sufficiently resolved (Huelsenbeck and Hillis 1993). Morphological analyses. Pholidosis was examined and morphometrics acquired for 79 individuals in four groups of Ph. mystaceus, including 20 specimens of nomina- tive subspecies Ph. m. mystaceus, seven specimens from Khorasan Province of Iran, 32 specimens of Ph. m. aurantiacocaudatus from Eastern Kazakhstan, and 20 specimens of Ph. m. galli from Middle Asia (Appendix 1). In order to take into account sexual dimorphism, males (n = 26) and females (n = 44) were analyzed separately. Morphological characteristics and the methods for their measurement are generally the same as in the study by Solovyeva et al. (2012). The following measurements and scalation counts were used: (1) snout-vent length (SVL); (2) tail length (TL); (3) SVL/TL ratio; (4) number of flat supralabials anterior to angular enlarged spine-like supralabial scales (SLbA); (5) total number of flat supralabials from tip of snout to insertion of cutaneous fold at mouth angle (SL); (6) relative length of the dark distal part of the tail to the total tail length (in ventral aspect, calculated as TL-black/TL ratio); (7) number of scales surround- ing subnasal from below (SSbNb); (8) subnasal in contact with medial side of supranasal (vs. subnasal not in contact with medial side of supranasal) (SbN-SpN); (9) supranasal edg- es nostril dorsally along the full length of nostril (vs. supranasal edges nostril dorsally along only half of nostril length) (SpN); (10) height of supranasal is less than or equal to height of subnasal (vs. height of supranasal exceeds height of subnasal) (hSpN SbN); (11) number of scale rows that separate subnasal and labial scales (SbN-L); (12) longitudinal row of white scales in supraorbital area outlined by continuous black lines (or intermitted) (WS8&BL); (13) number of small rows of scales between anterior (2d and 3d) inframandibulars and large rows of scales under infralabial scales — 1-2 or 2-3 (alMd-IL); (14) number of scales that underlay enlarged spiny scales on edge of cutaneous fold at mouth angle (SuSSCF); (15) number of small granular scales between posteriormost supralabial and insertion of cutaneous fold at mouth angle (pSL-CF); (16) number of flat infralabials anterior to an- gular enlarged spine-like infralabial scales (ILbA); (17) total number of infralabials from tip of snout to insertion of cutaneous fold at mouth angle (IL); (18) number of subdigital lamellae under toe III (SLID); (19) number of enlarged triangular scales on lateral fringes of toe II (FrIII); (20) number of subdigital lamellae under toe [V (SLIV); (21) number of enlarged triangular scales on lateral fringes of toe [V (FrIV). Characteristics 18-21 (SLI, Frill, SLIV, FrIV) were registered with no regard to the sex of the individual. Following standard measurements were additionally taken for holotype and paratypes: head height (HH); head length (HL, measured on ventral side from snout tip to gular fold); head width (HW, measured at broadest part of head excluding cutaneous folds); pileus width (PW). Measurements were taken using a digital caliper and rounded to the nearest 0.1 mm. Box-and-whiskers-plots and values of descriptive statistics were calculated using R (R Core Team, 2013). The Mann-Whitney test of independent series was used to determine the differences between the pairs of subspecies (with confidence level of p < 104 Evgeniya N. Solovyeva et al. / ZooKeys 748: 97-129 (2018) 0.05). Principal Components Analysis (PCA) was performed using R (R Core Team, 2013) to visualize morphological variation between Khorasan specimens of Ph. mysta- ceus and specimens from other populations. Results Sequence characteristics. The sequenced fragments from 31 Ph. mystaceus specimens were up to 654 b.p. in length, among which 577 sites were identified as conservative, 74 as variable and 59 as potentially parsimony-informative. Nucleotide frequencies were equal to: 30.2% (A), 27.5% (T/U), 27.9% (C), and 14.4% (G). The transition- transversion bias (R) was estimated to be 6.574 (all data given for in-group only). Phylogenetic analysis. The results of phylogenetic analysis are presented in Fig. 2. BI and ML yielded trees that show essentially similar topologies. All analyses reveal the presence of two reciprocally monophyletic clades within Ph. mystaceus. The first clade consists of Irainan P/. mystaceus ssp. from Khorasan Province (node support values are 1.0/86; hereafter given for BI PP/ ML BS; clade I on Fig. 2). The second clade includes all other P/. mystaceus populations from Middle and Central Asia and Caspian Region (1.0/99; clade II on Fig. 2). Further phylogenetic structure within the second clade of non-Iranian Ph. mystaceus is poorly resolved. Populations from the eastern part of the range including Eastern Kazakhstan and Xinjiang (China) that correspond to the Ph. m. “aurantiacocaudatus’ occupy basal position in the clade I, but are not monophyletic and fall into three poorly differentiated subclades: from the environs of Kapchagai (subclade A; 0.90/82), Ili River Valley (subclade B; from Zharkent to Xinjiang; 1.0/95), and the environs of Lake Balkhash (subclade C; 0.98/-) (see Fig. 2). Phylogenetic positions of two samples from Eastern Kazakhstan (ZMMU NAP-05510 and ZMMU R-12518-2) are not resolved. All other populations from Middle Asia (Kazakhstan and Uzbekistan — Fig. 2D ) and Caspian Region (Astrakhan Province, Russia — Fig. 2E) form a signifi- cantly monophyletic clade (1.0/95), which is deeply nested within the basal differentia- tion of East Kazakhstan Ph. m. “aurantiacocaudatus” clades (see Fig. 2), rendering the latter taxon paraphyletic. The Middle Asian — Caspian clade (D + E) corresponds to the nominative subspecies Ph. mystaceus mystaceus and also includes populations previously classified as Ph. mystaceus galli (Aral Sea Region, Uzbekistan and Tajikistan — Fig. 2D). Populations of Ph. mystaceus mystaceus and Ph. mystaceus “galli” are mixed with each other without any clear structure (see Fig. 2 D1, D2, E1, E2). Genetic distances. Uncorrected genetic p-distances between and within clades of Ph. mystaceus are shown in Table 2. The p-distances within the Middle Asian — Caspian clade of Ph. mystaceus, including comparisons between different lineages of Ph. m. “aurantiacocau- datus’ and between Ph. m. “aurantiacocaudatus” and Ph. m. mystaceus are quite low (0.55— 0.88% and 1.56—1.87%, respectively), which is less than intraspecific genetic distances for COI for some other species of Phrynocephalus (e.g. see Solovyeva et al. 2011 for Ph. helioscopus). However, p-distances between Ph. mystaceus ssp. from Khorasan Province and all other groups of P/. mystaceus are very high (6.84—7.28%), they even exceed interspecific Molecular and morphological differentiation of Secret Toad-headed agama... 105 ZMMU R-12328-1 Ph. melanurus ZMMU R-13169 Iran, Khorasan (3) ZMMU R-11913 Iran, Khorasan (1) BA/ML Ph. mystaceus 4l- 4/86] ZMMU R-12202 Iran, Khorasan (1) kh | ZMMU R-13011-1 Iran, Khorasan (1) orasanus ssp. Nn. ZMMU R-13011-2 Iran, Khorasan (1) lran, Khorasan ZMMU R-13009-1 Iran, Khorasan (2) 1/78 ZMMU R-13009-2 Iran, Khorasan (2) 0.9/82, RUHF-077-1 E Kazakhstan, N Kapchagai (12) if A f RuHF-077-2 E Kazakhstan, N Kapchagai (12) ZMMU R-12778 E Kazakhstan, Panfilov env. (14) NC021131 China, Ily river valley, Houchen (17) KC578685 China, lly river valley, Houchen (17) B Ph. mystaceus “aurantiacocaudatus” ZMMU R-12518-2 E Kazakhstan (13) Kazakhstan, Semirechye ZMMU R-14715-1 E Kazakhstan, S Balkhash (15) and lly river valley = + ZMMU R-14715-2 E Kazakhstan, S$ Balkhash (15) Cc ZMMU R-14715-3 E Kazakhstan, S Balkhash (15) ZMMU R-14715-4 E Kazakhstan, S Balkhash (15) NAP05510 E Kazakhstan, E Balkhash (16) ZMMU R-12252-1 Uzbekistan, Navoi (6) | | ZMMU R-12266 Uzbekistan, Qarakalpaqiston (8) D1 ZMMU R-12775 Uzbekistan, Qarakalpaqiston (10) 4/95|| ZMMU R-12457-2 Russia, Astrakhan region (4) | E1 ZMMU R-12261-1 Uzbekistan, Navoi (7) Ph. m. mystaceus RuHF-079-2 Kazakhstan, N Priaralye (5) ¥ H Fé, HET 0.97/-| L RuHF-079-1 Kazakhstan, N Priaralye (5) D2 (incl. Ph. m. “galli”) 193 ZMMU R-12517-2 Kazakhstan, N Priaralye (5) Russia, Uzbekistan, ZMMU R-12799 Tajikistan, Shaartuz (11) Tajikistan, West and ZMMU R-12772 Kazakhstan, Kzyl-Orda (9) Central Kazakhstan 0.99/78] | RuHF-072-1 Russia, Astrakhan region (4) RuHF-072-2 Russia, Astrakhan region (4) | 2 1100 | 7¥MU R-12457-3 Russia, Astrakhan region (4) KF691668 Trapelus sanguinolentus — 0.02 Figure 2. Bl-inferred dendrogram that illustrates the phylogenetic relationships of the Phrynocephalus mystaceus species complex based on the analysis of 654 b. p. fragment of CO/ gene (mtDNA). Numbers at the tree nodes show Bayesian Posterior Probabilities/ Maximum Likelihood Bootstrap Support. Only PP values higher than 0.90 and BS values higher than 75% are shown. COI sequence of Trapelus sanguino- lentus is used as an outgroup. Table 2. Uncorrected p-distances (percentage) between and within the groups of P/. mystaceus complex. Distances are shown under the diagonal row; standard error values are given above the diagonal row. P/. m. aurantiacocaudatus A corresponds to the population from N Kapchagai (RuHF-077); a — all specimens of Ph. m. aurantiacocaudatus, except for Ph. m. aurantiacocaudatus A, b — Ph. m. aurantiacocaudatus from southeast Pribalkhashye (Matay) (ZMMU R-14715), c — Ph. m. aurantiacocaudatus from Ili river valley, except for R-12518-2. Group b re 3 4 1. Ph. m. mystaceus (Including “Ph. m. gall?’) 0.41 0.96 2-a 7 0.94 2. Ph. m. “aurantiacocaudatus” 2-b 1.87 £02 2-c 1.65 1.03 3. Ph. m. “aurantiacocaudatus” A L.56 0.97 4. Ph. m. khorasanus ssp. n. 6.84 0.37 genetic distances for COJ gene reported for certain species of Phrynocephalus (Solovyeva et al. 2014). This data clearly suggest a deep divergence between Ph. mystaceus populations from Khorasan Province and populations from the rest of the range of the species. Morphology. Our study supports the results of previous researchers that indicated very high morphological variation in the absence of consistent morphological variation 106 Evgeniya N. Solovyeva et al. / ZooKeys 748: 97-129 (2018) patterns that could delimit recognized subspecies in Middle Asian populations of PA. mystaceus (Vel'dre 1964a, 1964b; Semenov and Shenbrot 1990). Most characteristics, including body size, were uninformative for distinguishing subspecies and local popu- lations of Ph. mystaceus. Only four morphological characteristics showed consistent differences between Iranian and Middle Asian/Caspian populations of Ph. mystaceus, including SLIV, FrIlI, SL, and TL-black/TL. Specifically, SLIV was lower in the popu- lation from Khorasan Razavi Province (N = 7) than in other subspecies of Ph. mystaceus (differences are significant; p = 0.000 for comparison with PA. h. “aurantiacocaudatus’, N = 32; p = 0.000 for comparison with PA. h. “galli”, N = 20; p = 0.087 for compari- son with Ph. m. mystaceus sensu stricto, N = 20; for measurement ranges see Table 3). FrIII was also significantly lower in Khorasan population (N = 7) than in Ph. m. mys- taceus sensu stricto (p = 0.000 N = 20). SL was also lower in Khorasan population (N = 7) than in other subspecies (p = 0.007 for comparison with Ph. m. “aurantiacocauda- tus’, N = 32; p = 0.001 for comparison with Ph. m. mystaceus sensu stricto, N = 20; p = 0.050 for comparison with Ph. m. “galli”, N = 20). Finally, the dark distal part of the tail (TL-black/TL) was relatively longer in the Khorasan population (differences are significant; p = 0.023, for comparison with Ph. m. “aurantiacocaudatus’, N = 32; p = 0.000 for comparison with Ph. m. mystaceus sensu stricto, N = 20; p = 0.001 for com- parison with Ph. m. “galli”, N = 20). Morphological comparison of four geographical population groups that correspond to the subspecies “systaceus sensu stricto’, “galli”, “aurantiacocaudatus’ and the “Khorasan population” for the diagnostic morphological characteristics mentioned above is given in Fig. 3. Other characteristics with p-values for pairwise comparisons <0.05 showed significant overlap of values between subspe- cies and cannot be reliably used in diagnostics; p-values for morphological characteris- tics for pairwise comparisons are summarized in Appendix 2. Standard measurements of Ph. mystaceus ssp. from Khorasan Province are presented in Table 4. Comparison of Khorasan P/. mystaceus ssp. population with other populations of Ph. mystaceus from Middle Asia and Caspian region (data for “mystaceus sensu stricto”, “gall”, “aurantiacocaudatus’ combined together) also demonstrated significant differ- ences for many traits with p<0.05 (Appendix 2), however, values for most of them were overlapping. The six of the characters with the minimal overlap were the following: SVL/TL, TL-black/TL, SL, ILbA, SLI, SLIV (see Fig. 4 for details). PCA showed differences between Khorasan population and other Ph. mystaceus populations, although these two groups are slightly overlapping with two Khorasan specimens falling into the Ph. m. mystaceus sensu lato area (Fig. 5). PCA failed to reveal any clear structuring within the Middle Asian / Caspian populations of Ph. mystaceus. Taxonomy MtDNA data strongly indicates the presence of two deeply divergent clades within P/. mystaceus: one from northeastern Iran, the other occupying the rest of the species range in Middle Asia (see Fig. 1). MtDNA divergence in CO/ gene fragments between these 107 Molecular and morphological differentiation of Secret Toad-headed agama... 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Solovyeva et al. / ZooKeys 748: 97-129 (2018) 24 26 28 30 32 34 12 14 16 18 10 0.40 0.45 12 13 14 15 16 17 18 19 0.35 0.36 0.38 0.40 0.42 0.44 0.46 D: males D: females [ | Ph. m. “aurantiacocaudatus” [| Ph. m. khorasanus ssp. n. [J Ph. m. mystaceus [] Ph. m. “galli” Figure 3. Statistically significant morphological differences between Ph. mystaceus khorasanus ssp. from Iran and other subspecies of Ph. mystaceus: A the number of subdigital lamellae on the toe IV (SLIV) B the number of enlarged triangular scales on the lateral fringe of the toe III (FrII) C the total number of supralabial scales (SL) D the relative length of the dark distal part of the tail to the total tail length (TL-black/TL). lineages is significant, 6.84—7.28% of substitutions, what corresponds to species-level divergence values in lizards, including the genus Phrynocephalus (Nagy et al. 2012; Nazarov et al. 2012, 2014; Solovyeva et al. 2012, 2014; Hartmann et al. 2013; Naz- arov & Poyarkov 2013; Amarasinghe et al. 2017; Orlova et al. 2017). According to Molecular and morphological differentiation of Secret Toad-headed agama... 111 22 34 24 26 28 30 A C: males C: females : a” D: males D: females E: males E: females [| Ph. m. mystaceus s.|. [| Ph. m. khorasanus ssp. n. Figure 4. Statistically significant morphological differences between Ph. mystaceus khorasanus ssp. from Iran and other Ph. mystaceus: A the number of subdigital lamellae on the toe HI (SLII) B the number of subdigital lamellae on the toe IV (SLIV) C the total number of supralabial scales (SL) D the relative length of the dark distal part of the tail to the total tail length (TL-black/TL) E number of flat infralabials anterior to the angular enlarged spine-like infralabial scales (IIbA). the data of Solovyeva et al. (2014), sequences of three other mtDNA genes of Iranian and Middle-Asian lineages of Ph. mystaceus are also deeply divergent: ND4 (6.6%), ND2 (8.0%) and cyt b (6.6%). Divergence time estimates (Solovyeva et al., 2018) suggest that the split between Iranian and Middle Asian Ph. mystaceus happened in the 112 Evgeniya N. Solovyeva et al. / ZooKeys 748: 97-129 (2018) Factor 2 -2 -3 -4 -3 -2 -1 0 1 2 3 Factor 1 eo Ph. m. khorasanus ssp. n. e Ph. m. mystaceus s.I. Figure 5. Principal Components Analysis (PCA) of 19 morphological traits (excluding SVL and TL). Pliocene, ca. 3.7 Ma (6.0—2.0 Ma). Thus, our data strongly indicate the presence of a deep-divergent mtDNA lineage of Ph. mystaceus in northeastern Iran which deserves taxonomic recognition. The question of the taxonomic status proposed for the Khorasan Ph. mystaceus populations, is, however, a matter of taste. On one hand, biogeographically the Kho- rasan Ph. mystaceus populations appear to be isolated from the main part of the spe- cies range in Middle Asia. The sands of the northeastern Iranian Plateau are located on much higher elevations (700-1000 m a.s.l.) than in the Middle Asia where Ph. m. mystaceus sensu lato occur (usually, 0-400 m a.s.l.), and are separated from the Caspian Basin by the Kopet-Dagh mountains, which has an estimated geologic uplift time of 5 Ma (Smit et al. 2013). The formation of Kopet-Dagh might be responsible for the initial split between the populations of Ph. mystaceus. The montane area of Kopet- Dagh lacking habitats suitable for P+. mystaceus, such as sand dunes, serves as a bar- rier preventing gene flow between the Middle Asian and the Khorasan populations. Geographic isolation resulted in deep molecular divergence might suggest that the full species status should be proposed for the Khorasan populations of Ph. mystaceus. Molecular and morphological differentiation of Secret Toad-headed agama... LS However, despite the significant molecular divergence, morphological differentia- tion between the Khorasan and Middle Asian linages of Ph. mystaceus is weak with only few morphological characteristics separating them. At the same time, individu- als of Ph. mystaceus from the vast range in the Caspian Region and Middle Asia are poorly differentiated both by morphometric characteristics (see Vel’dre 1964a, 1964b; Semenov and Shenbrot 1990; Golubev and Sattorov 1992) and by mtDNA (this pa- per). High morphological plasticity and variability are often recorded in specialized psammophilous groups of lizards (see Semenov and Shenbrot 1990). Both mtDNA and morphological data fail to resolve differentiation between the currently recognized non-Iranian subspecies of Ph. mystaceus: mystaceus sensu stricto, “galli” and “auranti- acocaudatus’. These subspecies are not supported as respective monophyletic groups in mtDNA analysis: the variation pattern is more likely clinal along the range from Xinjiang of China and Eastern Kazakhstan westwards to Middle Asia and Caspian Region. This suggests a recent dispersal of the non-Iranian Ph. mystaceus ancestor from a refugium in Eastern Kazakhstan westwards towards Caspian Basin. There is no morphological or mtDNA evidence for recognizing Ph. m. galli as a distinct subspecies; we therefore confirm the conclusions of Semenov and Shenbrot (1990) who regarded Ph. m. galli as a junior synonym of the nominative subspecies. The East Kazakhstan Ph. m. aurantiacocaudatus is paraphyletic with respect to Ph. m. mystaceus and is not supported as a valid taxon according to our mtDNA data. The only existing character distinguishing P/. m. aurantiacocaudatus from the representatives of other populations from Caspian Region and Middle Asia is the bright orange-red col- oration of the tail ventral surface in juvenile specimens. Unfortunately, this character cannot be verified on museum collections since orange tail coloration fades quickly after preservation. Analysis of morphometric and meristic characters could separate Ph. m. aurantiacocaudatus from the nominative form Ph. m. mystaceus. We conclude that the subspecific status of Ph. m. “aurantiacocaudatus” requires further justification. Our data show that the significant genetic differentiation of Khorasan Ph. mysta- ceus and presence of a number of stable diagnostic morphological characters warrants its recognition as a separate taxon. As noted above, genetic divergence between Ph. mystaceus from Khorasan and individuals from the rest of the species range is high, comparable or even exceeds the species-level genetic distances in Phrynocephalus (So- lovyeva et al. 2014). However, we tentatively refrain from proposing the full species status for this lineage, and suggest that, at least at the current stage of research, it should be recognized as a subspecies, for the following three reasons: (1) Due to matrilineal way of mtDNA inheritance and absence of recombination, even deep genetic divergence in mtDNA markers, does not guarantee reproductive isolation and should not serve as a sole reason for suggesting the full species status. (2) Morphologically, the Khorasan population is still quite similar to other P/. mystaceus populations and the revealed morphological differences are mostly quantita- tive, further morphological evidence is needed. (3) Our sampling from Khorasan Province of Iran is limited, further studies in northeastern Iran are needed to uncover new populations in the area between the 114 Evgeniya N. Solovyeva et al. / ZooKeys 748: 97-129 (2018) Ph. m. mystaceus range in Turkmenistan and the Khorasan population, genetic and morphological characterization of these populations is required. A recent analysis had shown that subspecies are getting more rarely proposed for the extant reptiles in the last 50 years (Uetz and Stylianou 2018), what is connected with a growing tendency to elevation of many subspecies to species and also with grow- ing prevalence of the phylogenetic species concept (Cracraft 1983), which does not recognize subspecies. However, we still consider subspecies to be a useful taxonomic category for reflecting geographic variation and evolutionary specificity in wide-ranged complexes of reptiles. Though taxonomic status of Middle Asian subspecies “gall?” and “aurantiacocaudatus’ is questionable, both mtDNA sequences and external morphol- ogy of the Khorasan population of Ph. mystaceus significantly differ from all other populations of this species. This allows us to describe it herein as a new subspecies: Phrynocephalus mystaceus khorasanus ssp. n. http://zoobank.org/6E926506-3D7A-4C99-BF64-A02C48157B5C Figs 6A, B; 7; 8; Table 4 Holotype. ZMMU R-11913 (adult female; field number NR-1191). Type locality. Iran, Khorasan historical area, Khorasan Razavi Province (ostan), environs of Gonabad, the right bank of the Kale-Shur River; sand dunes (see Fig. 9); N34°39', E58°43'; elevation 850 ma. s. |. Collected by Roman A. Nazarov and Mehdi Radjabizadeh on April 25, 2005. Paratypes. ZMMU R-13009 (one adult male with everted hemipenial structures, field number RAN 1723; and one adult female, field number RAN 1724) was collect- ed in Iran, Khorasan historical area, Khorasan Razavi Province, 20 km east of the town of Boshrouyeh (N33°54', E57°30'; elevation 864 m a. s. |.) by Dmitriy A. Bondarenko, Roman A. Nazarov, and Mehdi Radjabizadeh on May 05, 2009. The rest of paratypes were collected in the area close to the type locality. ZMMU R-13011 (one adult male with hemipenial structures, field number RAN 1947; and one subadult female, field number RAN 1948) was collected in Iran, Khorasan Razavi Province, 60 km north of the town of Gonabad, stabilized or semi-stabilized sands (N34°36', E58°14'; elevation 867 ma.s. |.) by Roman A. Nazarov, Rustam K. Berdiev, Vlad G. Starkov, and Mehdi Radjabizadeh on June 02, 2009. ZMMU R-13169 (subadult female) was collected in Iran, Khorasan Razavi Province, 30 km north of the town of Gonabad, on sandy massif on the right bank of the Kale-Shur river (N34°35', E58°43'; elevation 888 m a. s. 1.) by Roman A. Nazarov, Dmitriy A. Bondarenko, and Mehdi Radjabizadeh on May 10, 2010. ZMMU R-12202 (juvenile female with slightly orange lower surface of the tail, field number N-093) was collected in Iran, Khorasan Razavi Province, 60 km north of the town of Gonabad, on sands (N34°36', E58°44'; elevation 881 ma.s.l.) by Dmitriy A. Bondarenko on April 20, 2006. Diagnosis. A member of Ph. mystaceus species complex based on the following combination of morphological attributes: (1) a large-sized Phrynocephalus species with Molecular and morphological differentiation of Secret Toad-headed agama... 115 uh ee 99 Table A2. Mann-Whitney test of independent series: “aura” — Ph. m. “aurantiacocaudatus”, — Ph. m. khorasanus ssp. n., “myst” — Ph. m. mystaceus sensu stricto, “galli” — Ph. m. “galli”; for abbreviations, see Materials and Methods. Significant values of p < 0.05 are marked with bold and an asterisk. ALL Sexes aura-ir | it-myst | ir-galli | myst-aura| aura-galli | galli-myst| ir-all f-m 1.SVL 0.321 0.000* 0.013* 0.000* 0.008* 0.000* 0.005* | 0.007* 3. SVLITL 0.001* | 0.591 4. SLbA 0.014* 0.813 5. SL 0.003* 0.355 6. TL-black/TL 0.001* 0.527 7. SSbNb 0.140 0.201 8. SbN-SpN 0.570 0.169 9. SpN 0.457 | 0.022* 10. hSpN SbN 0.001% | 0.259 11. SbN-L 0.010* 0.589 0.097 0.000* 0.992 0.016* 0.144 0.234 13. aIMd-IL 0.030* | 0.091 14. SuSSCF 0.029 0.004* 09:12 0.164 0.000* 0.000* 0.174 0.839 16. ILbA 0.003* | 0.640 17. IL 0.023* 0.017* 0.059 0.645 0.013* 0.059 0.022* 0.588 19. Fefll 0.283 | 4, 20. SLIV 0.000* 0.087 0.000* 0.087 0.463 0.026* 0.000* Noe Molecular and morphological differentiation of Secret Toad-headed agama... «¥£0'0 | 1970 | | xZ10'0 | | 91Z'0 | O1Z'0 | 08y0 | AZT «700'0 149'0 dO-Isd C1 060'0 809'0 AOSSNS “FI «700'0 8750 TI-PWI “€T xI110°0 | VN 8ce'0 Z61'0 TAVSAM ‘ZI rIEO | 9900 799'0 C710 TNGS ‘TT x900°0 | 9S1°0 €18°0 €0I'0 Nas Ndsy ‘or Ecyv'0 | 6270 97S'0 «L10°0 O€€'0 Nds-Nqs ‘8 +8100 | ZZ0°0 | | 7000 | | FTO | | 1600 | TLLPPPI9-TL'9 x€70'O | TOTO <600°0 €90°0 «£40'0 1s ‘s x0F0'O | E80 9800 Ee ce VIS ‘¥ S¥0'0 | L¥6'0 | | 09z'0 | TL/TAS ‘€ «100°0 8Z€'0 $69'0 TL ‘Z ysAur pine ysAur yes pine et -I]yes -ysAur ea -1yes -eme -ysAur ccilinee SaTVWAA STTVI ‘ponunuoy "Ty ajqeL Evgeniya N. Solovyeva et al. / ZooKeys 748: 97-129 (2018) Figure 10. ZMMU R-6413, lectotype of Phrynocepahlus mystaceus galli Krassowsky, 1932 in preserva- tive: A dorsal view B ventral view C head in dorsal view D head in frontal view E head in lateral view F left foot in thenar view (photographs by E. N. Solovyeva). Molecular and morphological differentiation of Secret Toad-headed agama... Figure 11.ZMMU R-6412, holotype of Phrynocepahlus mystaceus aurantiacocaudatus Semenov & Shen- brot, 1990 in preservative: A dorsal view B ventral view C head in dorsal view D head in frontal view E head in lateral view F right foot in thenar view (photographs by E. N. Solovyeva).