Molecular and morphological differentiation of Secret Toad-headed agama, Phrynocephalus mystaceus, with the description of a new subspecies from Iran (Reptilia, Agamidae)

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 Ph. mystaceus by a combination of several morphological features. Molecular and morphological analyses do not support the validity of other Ph. mystaceus subspecies described from Middle Asia and Caspian basin. Geographic variations in the Ph. mystaceus species complex and the status of previously described subspecies were discussed.


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 Peninsula) through Middle Asia to Central Asia (northern and central China and Mongolia). This taxonomically complicated genus currently contains up to 32 species (Uetz and Hošek 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 variability (Ananjeva "1986(Ananjeva " " 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. mystaceus, 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 position 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 phylogeny based on the analysis of RAG1 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 Ph. mystaceus is a member of the "core" Phrynocephalus clade and is associated with Ph. axillaris (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 Ph. mystaceus. Krassowky (1932) was the first to split Ph. 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 (1964aVel'dre ( , 1964b suggested that it is impossible to distinguish geographical races within Ph. mystaceus due to its high morphological variability among populations. Consequently, Ananjeva (1987Ananjeva ( "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. 1987Ananjeva et al. "1986. Semenov and Shenbrot (1990) analyzed morphological and chromatic differentiation of Ph. mystaceus from "Semirechye" (an area east of lake Balkhash in Eastern Kazakhstan), and suggested that this area is inhabited by a distinct subspecies, Ph. mystaceus aurantiacocaudatus Semenov et Shenbrot, 1990, which differs from the Middle Asian subspecies Ph. m. galli by its bright orangered coloration of the ventral surface of the tail in young specimens (versus lemon-yellow coloration in other subspecies). However, Ph. mystaceus aurantiacocaudatus was synonymized with Ph. m. galli by Barabanov and Ananjeva (2007) without any discussion.
It is notable that all previous works on geographic variations of Ph. mystaceus omitted populations from the southernmost edge of its range, Iran and Afghanistan, from the analyses. Morphological characterization and analysis of distribution of Ph. mystaceus in Iran was carried out by Anderson (1999) and Molavi et al. (2014). Anderson (1999) Molavi et al. (2014), based on a study of seven specimens from Semnan Province, repeated earlier conclusions by Anderson (1999) and suggested that further investigation 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. mystaceus samples from Iran and Middle Asia (Solovyeva et al. 2014). Based on these results the Iranian population was tentatively indicated as a putative new subspecies Ph. mystaceus ssp. In the present study, we provide a detailed analysis of both morphologi-cal and genetic variation of Ph. mystaceus across its range and confirm deep differentiation 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.
PartitionFinder v1.0.1 (Lanfear et al. 2012) was used to estimate the optimal evolutionary models for Bayesian inference analysis. The preferred model for COI alignment 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 generations 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 Likelihood Weights (ELW).

Figure 1.
Geographical distribution of Phrynocephalus mystaceus and locations of the sites where the samples 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 Ananjeva, "1986" 1987 C Phrynocephalus mystaceus aurantiacocaudatus Semenov & Shenbrot, 1990D Phrynocephalus mystaceus galli Krassowsky, 1932; and E Ph. mystaceus khorasanus ssp. n. 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 nominative 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 surrounding 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 edges 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) (WS&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 (aIMd-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 angular 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 (SLIII); (19) number of enlarged triangular scales on lateral fringes of toe III (FrIII); (20) number of subdigital lamellae under toe IV (SLIV); (21) number of enlarged triangular scales on lateral fringes of toe IV (FrIV). Characteristics 18-21 (SLIII, FrIII, 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 ≤ 0.05). Principal Components Analysis (PCA) was performed using R (R Core Team, 2013) to visualize morphological variation between Khorasan specimens of Ph. mystaceus 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 transitiontransversion bias (R) was estimated to be 6.574 (all data given for in-group only). 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. "aurantiacocaudatus" 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 Ph. mystaceus are very high (6.84-7.28%), they even exceed interspecific genetic distances for COI 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 patterns that could delimit recognized subspecies in Middle Asian populations of Ph. mystaceus (Vel'dre 1964a(Vel'dre , 1964bSemenov and Shenbrot 1990). Most characteristics, including body size, were uninformative for distinguishing subspecies and local populations of Ph. mystaceus. Only four morphological characteristics showed consistent differences between Iranian and Middle Asian/Caspian populations of Ph. mystaceus, including SLIV, FrIII, SL, and TL-black/TL. Specifically, SLIV was lower in the population from Khorasan Razavi Province (N = 7) than in other subspecies of Ph. mystaceus (differences are significant; p = 0.000 for comparison with Ph. h. "aurantiacocaudatus", N = 32; p = 0.000 for comparison with Ph. h. "galli", N = 20; p = 0.087 for comparison 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. mystaceus 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. "aurantiacocaudatus", 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 comparison with Ph. m. "galli", N = 20). Morphological comparison of four geographical population groups that correspond to the subspecies "mystaceus 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 subspecies and cannot be reliably used in diagnostics; p-values for morphological characteristics 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 Ph. mystaceus ssp. population with other populations of Ph. mystaceus from Middle Asia and Caspian region (data for "mystaceus sensu stricto", "galli", "aurantiacocaudatus" combined together) also demonstrated significant differences 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, SLIII, 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 Ph. mystaceus: one from northeastern Iran, the other occupying the rest of the species range in Middle Asia (see Fig. 1). MtDNA divergence in COI gene fragments between these Table 3. Mean, standard deviation, and range of measurements (mm) of adult Ph. mystaceus ssp. For abbreviations, see Materials and methods. In SbN-SpN, SpN, hSpN-SbN, and WS&BL: 0 equals "no", 1 equals "yes".   0 equals "no", 1 equals "yes".   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 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.

Measurements
The question of the taxonomic status proposed for the Khorasan Ph. mystaceus populations, is, however, a matter of taste. On one hand, biogeographically the Khorasan Ph. mystaceus populations appear to be isolated from the main part of the species 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 Ph. mystaceus, such as sand dunes, serves as a barrier 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. However, despite the significant molecular divergence, morphological differentiation between the Khorasan and Middle Asian linages of Ph. mystaceus is weak with only few morphological characteristics separating them. At the same time, individuals of Ph. mystaceus from the vast range in the Caspian Region and Middle Asia are poorly differentiated both by morphometric characteristics (see Vel'dre 1964aVel'dre , 1964bSemenov and Shenbrot 1990;Golubev and Sattorov 1992) and by mtDNA (this paper). 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 "aurantiacocaudatus". 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 Ph. m. aurantiacocaudatus from the representatives of other populations from Caspian Region and Middle Asia is the bright orange-red coloration 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. mystaceus 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 (Solovyeva 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 Ph. mystaceus populations and the revealed morphological differences are mostly quantitative, 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 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 growing 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 "galli" and "aurantiacocaudatus" is questionable, both mtDNA sequences and external morphology 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   SVL up to 97.5 mm, tail shorter than SVL; (2) pair of cutaneous flaps present at mouth corners with numerous spiny scales along flap edges; (3) distinctly flattened body and tail; (4) toes with fringes formed by triangular scales; subdigital lamellae on toes III and IV with ridges. Phrynocephalus mystaceus khorasanus ssp. n. can be distinguished from the nominative subspecies of Ph. mystaceus by the following combination of two diagnostic morphological characteristics: (1) 24-27 lamellae on toe IV; (2) few supralabial scales (less than 14). In life, the new subspecies can be further distinguished from the nominative subspecies by the orange color of the lower surface of tail in young specimens (lemon to yellowish in Ph. m. mystaceus except the populations from Eastern Kazakhstan and western China, formerly described as Ph. m. aurantiacocaudatus). MtDNA sequences of Phrynocephalus mystaceus khorasanus ssp. n. are markedly distinct from those in all other populations of Ph. mystaceus with sequence divergence in the range of 6.84-7.28% between them. The new subspecies is notably smaller than the representatives of southern populations of Ph. m. mystaceus from Uzbekistan and Turkmenistan, formerly described as Ph. m. galli, which can reach SVL up to 122.7 mm (Anderson 1999), whereas for Iranian population Anderson (1999) reported the largest specimen of Ph. mystaceus to have SVL up to 77.7 mm. SVL in the largest specimen in our sampling reached 86.0 mm, while Molavi et al. (2014) recorded a specimen with SVL of 97.5 from Semnan Province.
Etymology. The name of the new subspecies khorasanus is a Latinized toponymic adjective, derived from Khorasan, the name of the historic area and a Khorasan Razavi Province in the northeast Iran, where the new subspecies was found. We suggest the "Khorasan Secret Toad-headed Agama" as a common name in English.
Description of holotype. Medium-sized agamid lizard, adult female, specimen in good state of preservation; body dissected on ventral side along the midline of belly (dissection ca. 20 cm in length). Measurements and counts of the holotype are presented in Table 4.
Head large, rounded, distinctly wider than neck region (see Fig. 7A); body and tail notably flattened. Snout abruptly blunt, head almost vertical in profile view (see Fig. 7E), nostrils invisible dorsally (see Fig. 7C). Nasals separated from each other by single scale (see Fig. 7D). Dorsal surface of head with distinct pileus consisting of small slightly keeled scales; ca. 30 scales across the pileus. Pineal scale separated from nasals by 13 smaller scales; scales covering orbital area somewhat smaller than those on frontal surface of head; occipital scales not enlarged. Five scales contacting subnasal ventrally (see Fig. 7D). Subnasal scale not in contact with inner (medial) side of supranasal. Supralabials separated from subnasal scale by 6 rows of small granular scales (see Fig. 7D). Pair of skin-folds form characteristic ear-shaped flaps in mouth corners, edges of each flap with enlarged conical scales, two groups of similarly enlarged conical scales on each side of head posterior to the mouth angle at tympanal area (see Fig. 7E). Supralabial scales anterior to cutaneous fold at mouth angle 11/12 (hereafter data for symmetrical characteristics is given in Right/Left order); 9/9 of anterior supralabials notably flattened, 2/3 posterior supralabials conical-shaped; supralabials separated from small granular scales of lower eyelid by 3/4 rows of scales, ventral row of these scales almost the same size as supralabials (see Fig. 7E). Single small scale between the posteriormost supralabial and insertion of cutaneous fold at mouth angle. Infralabial scales anterior to cutaneous fold -6/6, 3/3 of anterior infralabials notably flattened, posterior infralabials cone-shaped. Posterior corner of eye and insertion of cutaneous fold at mouth angle separated by row of three enlarged flat scales (see Fig. 7E). Vertebral scales not enlarged. Scales at middle of dorsum slightly bigger than scales on dorsolateral and lateral surfaces of body. Dorsal scales with weak keels, becoming cone-shaped laterally, forming almost triangular spines on the flanks. Notably enlarged spiny scale (about four times the size of adjacent scales) on each side of thorax behind maxilla, two groups of enlarged spiny scales on each lateral surface of neck region (see Fig. 7E). Tail notably flattened along its whole length. Scales on dorsal surface of tail and on ventral surface of tail posterior half notably keeled; scales on lateral sides of tail with well-pronounced spines. Limbs comparatively long: hindlimb length greater than distance from cloaca to gular fold. Toe IV bearing a single row of subdigital lamellae, each with a well-pronounced ridge on its volar surface; lateral sides of toe IV with two rows of enlarged triangular scales that form distinct serrated fringe (see Fig. 7F). Similar crests present on lateral surfaces of toe III, triangular scales on toe III notably smaller compared to those on toe IV (see Fig. 7F). Number of lamellae on toe IV 24/24, on toe III 16/16; number of enlarged triangular scales on toe IV 20/20, on toe III 9/9.
Color of holotype in life. In life dorsum sandy-beige; with numerous small black and white dots and reticulations; row of three pairs of irregular-shaped larger dark blotches on each side of vertebral line; ventral surfaces of body, limbs and proximal part of tail white; ventral surface of tail tip black, chin and throat with gray reticulations, chest with blackish longitudinal blotch. Ten brownish transverse bars (wider than interspaces) on dorsal surface of tail, faint at tail basis, get more distinct towards tail tip. Internal surfaces of mouth angle cutaneous flaps in life are pinkish, and may become red when animal displays a threatening posture.
Color of holotype in preservative. In preservative, numerous dark spots and mottling are distinct on dull sandy-gray background color of dorsum. They form vermiculate patterns ca. 1-2 scales wide. On lateral parts of dorsum these lines form 6-7 indistinct dark transverse bands. Ten dark transverse bars on dorsal side of tail are welldistinct (Fig. 7A). Three posterior dark bars have a distinct light-beige longitudinal line between them along midline of tail. Tail ventral surface light yellowish-white. Ventral surface of head with distinctive dark greyish marbling (Fig. 7A). Distinct triangular longitudinal black spot in the middle of chest area resembling a "necktie", ca. 8.8 mm in length. Black coloration of distal part of ventral surface of tail 24 mm in length.
Paratype variation. Variations of morphological characteristics in the type series are shown in Table 4 and in Fig. 8. In general, morphology of paratypes corresponds well to morphology of the holotype. SVL of new subspecies varies in range of 85.0-86.0 mm in two males, and in range of 54.0-70.0 in five females; tail length 76.0 mm in males, 51.0-67.0 mm in females; tail comparatively shorter in male specimens (SVL/TL ratio 1.12-1.13) than in females (SVL/TL ratio 1.00-1.06); however, the sample size is too small to detect significant differences. Length of dark distal part of ventral surface of tail varies from 20 to 27 mm. Number of subdigital lamellae on toe III varies from 17 to 20, from 25 to 28 on toe IV. Number of enlarged triangular scales of lateral fringes on toe III from 7 to 11, on toe IV from 18 to 21. Number of flattened anterior supralabials 6-11, total number of supralabials (to insertion of cutaneous fold at mouth angle) varies from 10 to 15. Number of small scales ventrally in contact with subnasal scale 3-6. Subnasal scale in all paratypes (except one specimen ZMMU R-13009) touches supranasal along medial edge of latter. In nearly all paratypes supralabials are separated from subnasal by five rows of small scales (only in ZMMU R-13009 by 4/5 rows of small scales). In most specimens, there is one small scale between last supralabial and insertion of cutaneous fold at mouth angle (specimen ZMMU R-13011 has two scales, ZMMU R-13169 lacks such scales). Number of flat anterior infralabials varies from 2 to 4, total number of infralabials to insertion of cutaneous fold at mouth angle varies from 5 to 7 (only ZMMU R-13009 has 3/3 infralabials). Number of black irregularly shaped spots on dorsum also may vary: from 4 to 6 pairs of black spots on each side of vertebral line (see Fig. 8A).
We were unable to detect sexual dimorphism in morphometric and meristic characteristics of Ph. mystaceus khorasanus ssp. n., however our sample size (N = 7) was too small. Molavi et al. (2014), who also examined seven specimens of both sexes from Semnan Province, was also unable to detect sexual dimorphism in morphological features in their sample.
Distribution. To date, the new subspecies is known from two major localities in southwestern part of Khorasan Razavi Province (environs of the towns of Gonabad and Boshrouyeh, this study) and from a single locality in the easternmost part of Semnan Province of Iran (Ahmad Abad village, Molavi 2014). The record from the environs of the town of Boshrouyeh appears to be the southernmost known locality for Ph. mystaceus complex known to date. The three records of Ph. mystaceus by Anderson (1999) from the northern part of Khorasan Razavi Province, North Khorasan and Golestan provinces are all located along the border with Turkmenistan. These populations most likely correspond to Ph. m. mystaceus rather than to Ph. mystaceus khorasanus ssp. n. as they are close to the range of the nominative form and there are no biogeographic barriers that separate these populations. On the contrary, localities in Khorasan Razavi and Semnan provinces are situated on different elevations and sand massifs are isolated from the range of Ph. m. mystaceus by at least 200 km of habitats unsuitable for Ph. mystaceus. We anticipate new records of the new subspecies in sandy areas of Khorasan Razavi, Semnan and, possibly, northern part of Yazd and South Khorasan provinces.
Habitat. Ph. mystaceus khorasanus ssp. n. inhabits sandy areas with sparse vegetation in northeast Iran at comparatively higher altitudes, than other Ph. mystaceus subspecies. The usual habitat is represented by dunes of loose sands and semi-stabilized dunes with rare grass, occasional bushes of Haloxylon sp. and Tamarix sp. and large open sandy areas (Fig. 9). The areas inhabited by the new subspecies receive almost no rainfall during the year. In the town of Gonabad the average annual temperature is 17.3 °C, the average temperature in July reaches 29.2 °C, the average temperature in January is 4.8 °C; In Boshrouyeh the average annual temperature is 19.7 °C, the average temperature in July is 31.9 °C, the average temperature in January is 6.6 °C. (http://www.climate-data.org).
Lizards burrow in sand, digging short tunnels and chambers; they can quickly dig into sand by rapid lateral movements of the body (Anderson, 1999).
Comparisons with other subspecies. Comparisons of the new subspecies from Khorasan Razavi and Semnan provinces of Iran with the nominative subspecies Ph. m. mystaceus sensu lato from Middle Asia, Caspian basin, and westernmost Xinjiang (China) are summarized below. In preservative, the new subspecies can be differentiated from specimens of Ph. m. mystaceus by the following combination of morphological attributes: lower number of subdigital lamellae on the IVth toe  We do not recognize Ph. m. galli as a separate subspecies due to the absence of stable genetic and morphological differences of this subspecies from Ph. m. mystaceus (see above). The Phrynocephalus mystaceus dagestanica form from Daghestan (Ananjeva, "1986(Ananjeva, " " 1987 is very close to the populations from the Volga River basin and was considered a synonym of Ph. m. mystaceus by several authors (Semenov and Shenbrot 1990;Barabanov and Ananjeva 2007). Our molecular and morphometric data do not support monophyly or significant differentiation of Ph. m. aurantiacocaudatus from Eastern Kazakhstan and western China. The only stable difference between this population and Ph. m. mystaceus sensu stricto is the tail coloration in juveniles. We consider that additional genetic and morphological data is needed to clarify taxonomic status of East Kazakhstan Ph. mystaceus populations.
Discussion. Our study indicates deep genetic divergence between Iranian populations of Ph. m. khorasanus ssp. n. and the rest of the populations within the range of the species. However, morphological differentiation within Ph. mystaceus complex is less clear with only a few morphological characteristics that reliably separate these two lineages. Differentiation pattern for the mtDNA COI gene within the Middle Asian and Caspian populations of Ph. mystaceus complex suggests that East Kazakhstan was populated by Ph. mystaceus earlier than the rest of Middle Asia. After that, a dispersal process from the east to the west likely took place. Morphologically different populations of Ph. mystaceus across Middle Asia present considerable amount of variation both in body size and in such morphological features as the relative size of cutaneous flaps in the mouth angles, relative tail length, etc. This high morphological plasticity may be connected with psam-mophilous biology of this species, as it was suggested by previous researchers (Vel 'dre 1964a, 1964bSemenov and Shenbrot 1990;Golubev and Sattorov 1992).
The data of phylogenetic analyses in the present paper clearly indicates that the whole territory of Middle Asia, including westernmost China and Caspian region, is inhabited by a single poorly differentiated mtDNA lineage. Golubev and Sattorov (1992) argued that coloration of the ventral tail surface in juveniles of Ph. mystaceus is also subject to high variation, and "orange-" and "yellow-tailed" specimens can be occasionally recorded within the same population, thus suggesting that subspecies within Ph. mystaceus should not be recognized. Our mtDNA genealogy indicates that both Ph. m. "galli" and Ph. m. "aurantiacocaudatus" do not form a respective monophyletic units and are genetically indistinguishable or very close the nominative subspecies P. m. mystaceus sensu stricto (p-distance 1.65-1.87% in case of East Kazakhstan populations).
On the contrary, the Khorasan population described herein as Ph. m. khorasanus shows very deep genetic divergence in mtDNA which is comparable to the specieslevel divergence in Phrynocephalus, but is only moderately differentiated morphologically. Indeed, previous research on four mtDNA genes also showed significant differentiation between Ph. mystaceus from Khorasan and Ph. m. mystaceus (p-distances: COI -7.18%; ND4 -6.6%; ND2 -8.0%; and cyt b -6.6%) (see Solovyeva et al. 2014). According to our unpublished data on molecular dating of 4 mtDNA genes these two forms diverged during Pliocene about 3.7 Ma (Solovyeva et al., 2018). Further studies are required to verify the taxonomic status of Ph. m. khorasanus ssp. n., including morphological examination of larger samples and molecular analysis of the nuclear DNA markers in order to check the presence of possible isolation between the Iranian and Middle Asian forms of Ph. mystaceus. The new subspecies inhabits sand dunes in the northeastern Iran; this desert area is separated from the range of Ph. m. mystaceus by Kopet-Dagh Mountain Ridge making the possibility of gene flow between these populations quite low. However, the taxonomic status of Ph. mystaceus populations reported by Anderson (1999) from northern Iran (northern parts of Golestan, North Khorasan and Khorasan Razavi provinces) is unclear and require verification. Additional fieldwork in northern Iran, western Afghanistan, and southern Middle Asia is required to recover new populations of Ph. mystaceus complex. Further progress in understanding of the phylogenetic relationships within Ph. mystaceus complex might lead to reconsideration of the taxonomic status of the Khorasan population as a full species. edev, Anna A. Bannikova and Alexander Y. Presnyakov. For permission to study specimens under her care and support, we thank Valentina F. Orlova (ZMMU). We are sincerely grateful to Kai Wang and one anonymous reviewer for their useful comments on the earlier version of the manuscript. Table A2.