Research Article |
Corresponding author: Evgeniya N. Solovyeva ( anolis@yandex.ru ) Corresponding author: Nikolay A. Poyarkov Jr. ( n.poyarkov111@gmail.com ) Academic editor: Johannes Penner
© 2018 Evgeniya N. Solovyeva, Evgeniy N. Dunayev, Roman A. Nazarov, Mehdi Rajabizadeh, Nikolay A. Poyarkov Jr..
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.
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
|
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.
Khorasan, molecular phylogenetics, morphology, phylogeography, Phrynocephalus mystaceus khorasanus , taxonomy
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 (
Phrynocephalus mystaceus was shown to have a high level of anatomical variability (
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
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 (
In summary, three subspecies of Phrynocephalus mystaceus are recognized in recent literature (see
1. Ph. m. mystaceus (Pallas, 1776), that inhabits eastern Ciscaucasia (eastern part of Chechen Republic, Daghestan, Kalmykia), Caspian region (southern part of Astrakhan Region, east of the Volga-Ural Sands; introduced to the Apsheron Peninsula, Azerbaijan) and northwestern Kazakhstan (
2. Ph. mystaceus galli Krassowsky, 1932, that inhabits Transcaspian Region and Middle Asia from Turkmenistan, Uzbekistan, Kazakhstan, to northeastern and eastern Iran and adjacent areas of Afghanistan (
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 Ph. mystaceus galli by
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
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 (
Sampling. Historical collections of the Zoological Museum of Lomonosov Moscow State University (
Molecular analyses. Mitochondrial DNA COI gene (cytochrome oxidase c subunit 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 standard phenol-chloroform extraction protocol followed by ethanol precipitation of DNA (
Sequences were aligned using Seqman 5.06 and checked using BioEdit Sequence Alignment Editor 7.1.3.0 (
List of the samples used in molecular analyses. Locality numbers correspond to those in Figure
Voucher No | Subspecies | Locality | GenBank No |
---|---|---|---|
|
Ph. mystaceus khorasanus ssp. n. | Iran, Khorasan Razavi Prov., Gonabad (1) | MF567983 |
|
Ph. mystaceus khorasanus ssp. n. | Iran, Khorasan Razavi Prov., Boshrue (2) | MF567989 |
|
Ph. mystaceus khorasanus ssp. n. | Iran, Khorasan Razavi Prov., Boshrue (2) | KF691714 |
|
Ph. mystaceus khorasanus ssp. n. | Iran, Khorasan Razavi Prov., Gonabad (1) | MF567987 |
|
Ph. mystaceus khorasanus ssp. n. | Iran, Khorasan Razavi Prov., Gonabad (1) | MF567988 |
|
Ph. mystaceus khorasanus ssp. n. | Iran, Khorasan Razavi Prov., Gonabad (1) | MF567975 |
|
Ph. mystaceus khorasanus ssp. n. | Iran, Khorasan Razavi Prov., 30 km N Gonabad (3) | MF567974 |
RuHF-072-1 | Ph. mystaceus mystaceus | Russia, Astrakhan Prov., Dosang (4) | MF567968 |
RuHF-072-2 | Ph. mystaceus mystaceus | Russia, Astrakhan Prov., Dosang (4) | MF567969 |
|
Ph. mystaceus mystaceus | Russia, Astrakhan Prov., Dosang (4) | MF567990 |
|
Ph. mystaceus mystaceus | Russia, Astrakhan Prov., Dosang (4) | MF567986 |
RuHF-079-1 | Ph. mystaceus galli | Kazakhstan, N Priaralye, S border of Malye Barsuki sands (5) | MF567971 |
RuHF-079-2 | Ph. mystaceus galli | Kazakhstan, N Priaralye, S border of Malye Barsuki sands (5) | MF567970 |
|
Ph. mystaceus galli | Kazakhstan, N Priaralye, S border of Malye Barsuki sands (5) | MF567985 |
|
Ph. mystaceus galli | Kazakhstan, Aralsk (6) | MF567982 |
|
Ph. mystaceus galli | Uzbekistan, Qarakalpaqiston Republic (7) | MF567981 |
|
Ph. mystaceus galli | Uzbekistan, Qarakalpaqiston Republic, Chukurkak (8) | MF567978 |
|
Ph. mystaceus galli | Uzbekistan, Navoi Prov., Terankuduk (9) | MF567977 |
|
Ph. mystaceus galli | Uzbekistan, Navoi Prov., Yamankum desert (10) | KF691713 |
|
Ph. mystaceus galli | Tajikistan, Shaartuz (11) | MF567979 |
RuHF-077-1 | Ph. mystaceus aurantiacocaudatus | E Kazakhstan, N Kapchagai Reservoir (12) | MF567972 |
RuHF-077-2 | Ph. mystaceus aurantiacocaudatus | E Kazakhstan, N Kapchagai Reservoir (12) | MF567973 |
|
Ph. mystaceus aurantiacocaudatus | SE Kazakhstan, left bank of Ili River,125 km of the road Almaty-Bakanas (13) | MF567984 |
|
Ph. mystaceus aurantiacocaudatus | Kazakhstan, Pidzhim env. (14) | MF567980 |
|
Ph. mystaceus aurantiacocaudatus | Kazakhstan, S Balkhash lake, N of Matay (15) | MF567991 |
|
Ph. mystaceus aurantiacocaudatus | Kazakhstan, S Balkhash lake, N of Matay (15) | MF567992 |
|
Ph. mystaceus aurantiacocaudatus | Kazakhstan, S Balkhash lake, N of Matay (15) | MF567993 |
|
Ph. mystaceus aurantiacocaudatus | Kazakhstan, S Balkhash lake, N of Matay (15) | MF567994 |
|
Ph. mystaceus aurantiacocaudatus | Kazakhstan, E Balkhash lake, environs of Kabanbay (16) | MF567995 |
No voucher number | Ph. mystaceus aurantiacocaudatus | China, Ili River valley, Huocheng (17) | NC021131 |
Mean uncorrected p-distances and sequences characteristics were calculated using MEGA 6 (
PartitionFinder v1.0.1 (
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).
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 (
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
Morphological characteristics and the methods for their measurement are generally the same as in the study by
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.
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.
BI-inferred dendrogram that illustrates the phylogenetic relationships of the Phrynocephalus mystaceus species complex based on the analysis of 654 b. p. fragment of COI 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 sanguinolentus is used as an outgroup.
Genetic distances. Uncorrected genetic p-distances between and within clades of Ph. mystaceus are shown in Table
Uncorrected p-distances (percentage) between and within the groups of Ph. mystaceus complex. Distances are shown under the diagonal row; standard error values are given above the diagonal row. Ph. 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) (
Group | 1 | 2 | 3 | 4 | |||
---|---|---|---|---|---|---|---|
2-a | 2-b | 2-c | |||||
1. Ph. m. mystaceus [Including “Ph. m. galli”) | 0.97 | 0.4 | 0.47 | 0.43 | 0.41 | 0.96 | |
2. Ph. m. “aurantiacocaudatus” | 2-a | 1.7 | 0.63 | – | – | 0.22 | 0.94 |
2-b | 1.87 | – | 0.16 | 0.34 | 0.32 | 1.02 | |
2-c | 1.65 | – | 0.88 | 0.27 | 0.25 | 1.03 | |
3. Ph. m. “aurantiacocaudatus” A | 1.56 | 0.66 | 0.88 | 0.55 | 0.15 | 0.97 | |
4. Ph. m. khorasanus ssp. n. | 7.28 | 7.18 | 7.24 | 7.17 | 6.84 | 0.37 |
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 (
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 (FrIII) 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).
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
Statistically significant morphological differences between Ph. mystaceus khorasanus ssp. from Iran and other Ph. mystaceus: A the number of subdigital lamellae on the toe III (SLIII) 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 (IlbA).
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.
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”.
Subspecies | A. mystaceus s. str. | B. “galli” | C. “aurantiacocaudatus” |
D. mystaceus s. l. (A+B+C) |
E. khorasanus ssp. n. | |||||
---|---|---|---|---|---|---|---|---|---|---|
Measure-ments |
m
N = 9 |
f
N = 11 |
m
N = 7 |
f
N = 13 |
m
N = 8 |
f
N = 15 |
m
N = 26 |
f
N = 44 |
m
N = 2 |
f
N = 5 |
SVL | 10.13±1.06 (7.9–11.2) |
9.26±0.89 (6.3–10.5) |
8.41±1.00 (6.4–9.5) |
8.18±1.02 (6.3–10) |
8.10±1.23 (6.5–10.5) |
6.97±1.31 (5.5–10) |
9.01±1.42 (6.4–11.2) |
8.19±1.41 (6.3–10.5) |
8.55±0.07 (8.5–8.6) |
5.96±0.65 (5.4–7) |
TL | 11.12±0.97 (9.3–11.2) |
10.14±0.87 (7.3–11.2) |
8.53±0.99 (6.4–9.7) |
7.28±1.03 (6.1–10.6) |
8.30±1.50 (7–11.5) |
7.27±1.28 (6–10.5) |
9.50±1.74 (6.4–11.5) |
8.49±1.63 (6–11.2) |
7.60±0.00 (7.6) |
5.8±0.61 (5.1–6.7) |
SVL/TL | 0.92±0.05 (0.85–1.00) |
0.91±0.04 (0.85–0.97 |
0.97±0.05 0.93–1.06 |
1.02±0.05 (0.83–1.10 |
0.98±0.04 (0.91–1.03) |
0.96±0.04 (0.88–1.03) |
0.96±0.05 (0.85–1.06) |
0.97±0.07 (0.83–1.10) |
1.13±0,01 (1.12–1.13) |
1.03±0.03 (1.00–1.06) |
SLbA | 12.11±0.60 (11–13) |
11.6±3.03 (7–17) |
10.14±1.35 (8–12) |
11.25±1.95 (7–15) |
10.5±1.77 (8–14) |
11±1.63 (7–13) |
10.93±1.55 (8–14) |
11.06±2.27 (7–17) |
8.50±3.53 (6–11) |
9.00±1.73 (8–11) |
SL | 15.78±1.39 (14–18) |
16.11±1.90 (13–19) |
13±1.63 (10–15) |
14.75±1.88 (12–19) |
14.75±1.49 (13–17) |
14.69±1.54 (12–18) |
14.83±1.76 (10–18) |
14.94±1.77 (12–19) |
11.5±2.12 (10–13) |
12.67±1.15 (12–14) |
TL-black/TL | 0.42±0.02 (0.38–0.45) |
0.44±0.03 (0.39–0.48) |
0.44±0.02 (0.41–0.47) |
0.41±0.04 (0.33–0.47) |
0.39±0.02 (0.35–0.41) |
0.39±0.03 (0.32–0.45) |
0.42±0.03 (0.35–0.47) |
0.41±0.04 (0.32–0.48) |
0.36±0.00 (0.36) |
0.39±0.01 (0.37–0.40) |
SSbNb | 5±0.71 (4–6) |
4.8±0.42 (4–5) |
6.14±0.69 (5–7) |
5.81±1.38 (3–8) |
5.75±1.(8 4–8) |
5.13±1.09 (4–7) |
5.57±0.94 (4–8) |
5.38±1.16 (3–8) |
5±1.41 (4–6) |
4.33±1.53 (3–6) |
SbN-SpN | 0.44±0.53 (0–1) |
0.40±0.52 (0–1) |
0.71±0.49 (0–1) |
0.56±0.51 (0–1) |
0.38±0.52 (0–1) |
0.13±0.34 (0–1) |
0.43±0.50 (0–1) |
0.38±0.49 (0–1) |
0.50±0.71 (0–1) |
0±0.00 (0) |
SpN | 0.33±0.50 (0–1) |
0.50±0.53 (0–1) |
0.71±0.49 (0–1) |
0.88±0.34 (0–1) |
0.25±0.46 (0–1) |
0.56±0.51 (0–1) |
0.50±0.50 (0–1) |
0.63±0.49 (0–1) |
0±0.00 (0) |
0.67±0.58 (0–1) |
hSpN-SbN | 0.78±0.44 (0–1) |
0.90±0.32 (0–1) |
0.57±0.53 (0–1) |
0.44±0.51 (0–1) |
0.88±0.35 (0–1) |
0.56±0.51 (0–1) |
0.70±0.47 (0–1) |
0.65±0.48 (0–1) |
0±0.00 (0) |
0±0.00 (0) |
SbN-L | 5.22±1.39 (3–8) |
5.30±0.79 (4.5–7) |
4.14±1.07 (3–6) |
4.78±1.22 (3–7 |
3.88±0.64 3–5) |
4.34±0.60 (3–5 |
4.50±1.14 (3–8) |
4.76±1.03 (3–7) |
5.00±0.00 (5) |
5±0.00 (5) |
WS&BL | 1±0.00 (1) |
1±0.00 (1) |
1±0.00 (1) |
0.94±0.25 (0–1 |
0.75±0.46 0–1) |
0.75±0.41 (0–1) |
0.90±0.31 (0–1) |
0.88±0.32 (0–1) |
0.5±0.71 (0–1) |
0.33±0.58 (0–1) |
aIMd-IL | 2±0.87 (1–3) |
1.95±1.30 (0–4) |
2±0.58 (1–3) |
1.59±0.84 (0–3 |
1.88±0.64 1–3 |
1.31±0.48 (1–2) |
1.80±0.71 (1–3) |
1.57±0.83 (0–4) |
1.5±0.71 (1–2) |
2.33±0.29 (2–2.5) |
SuSSCF | 2.4±0.53 (2–3) |
2.60±0.52 (2–3) |
1.79±0.49 (1–2.5) |
1.72±0.55 (1–3) |
2.31±0.70 (1–3) |
2.41±0.46 (2–3) |
1.98±0.79 (1–3) |
2.14±0.63 (1–3) |
2±0.71 (1.5–2.5) |
1.67±0.58 (1–2) |
pSL-CF | 1.56±0.53 (1–2) |
1.60±0.70 (0–2) |
2.14±0.90 (1–3) |
2.19±0.91 (1–4) |
1.25±0.71 (0–2) |
1.36±0.5 (1–2) |
1.77±0.77 (0–2) |
1.83±0.75 (0–4) |
1.2±0.71 (1–2) |
0.67±0.58 (0–1) |
ILbA | 4.17±1.03 (3–6) |
4±1.38 (1–6) |
5.10±1.20 (3–7) |
5.29±1.07 (2.5–7) |
4.5±0.53 (4–5) |
5.07±1.62 (2–7) |
4.57±1.04 (3–7) |
4.70±1.50 (1–7) |
2.50±0.71 (2–3) |
3.4±0.55 (3–4) |
IL | 6.5±1.45 (3–9) |
7.15±1.04 (5–9) |
7.50±1.51 (5–10) |
7.47±0.92 (6–9) |
6.75±0.71 (6–7) |
6.6±1.24 (3–8) |
6.9±1.35 (3–10) |
7.08±1.13 (3–9) |
6±1.41 (5–7) |
5.2±2.05 (3–7) |
SLIII | 21.77±1.61 (19–25) |
20.27±1.68 (17–29) |
20.44±2.05 (16–24) |
20.79±1.89 (16–29) |
18.14±1.45 (16–20) |
|||||
FrIII | 9.97±0.85 (9–12) |
9.87±0.97 (8–12) |
8.31±0.86 (7–11) |
9.35±1.18 (7–12) |
8.86±1.35 (7–11) |
|||||
SLIV | 30.93±1.95 (27–35) |
29.67±2.11 (25–35) |
30.03±1.93 (25–33) |
30.15±1.99 (25–35) |
25.71±1.25 (24–27) |
|||||
FrIV | 19.97±1.87 (16–25) |
18.87±1.70 (16–23) |
16±1.87 (13–21) |
18.22±2.48 (13–25) |
18.86±1.21 (18–21) |
Measurements (mm) of adult Ph. mystaceus khorasanus ssp. n. For abbreviations, see Materials and methods. In SbN-SpN, SpN, hSpN-SbN and WS&BL: 0 equals “no”, 1 equals “yes”.
Measurements |
Specimen ID |
SVL | TL | SVL/TL | HL | HH | HW | PW | SLbA | SL | TL-black/TL | SSbNb | SbN-SpN |
Specimen ID |
SpN | hSpN- SbN | SbN-L | WS&BL | aIMd-IL | SuSSCF | pSL-CF | ILbA | IL | SLIII | FrIII | SLIV | FrIV |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Males (N = 2) | R-13011-1 | 85.0 | 76.0 | 1.12 | 19.8 | 11.0 | 21.7 | 13.8 | 6 | 10 | 0.355 | 6 | 0 | R-13011-1 | 0 | 0 | 5 | 1 | 2 | 1.5 | 2 | 3 | 7 | 18 | 10 | 26 | 21 |
R-13009-1 | 86.0 | 76.0 | 1.13 | 19.3 | 11.8 | 18.3 | 14.3 | 11 | 13 | 0.355 | 4 | 1 | R-13009-1 | 0 | 0 | 5 | 0 | 1 | 2.5 | 1 | 2 | 5 | 20 | 11 | 26 | 18 | |
Range | 85.0–86.0 | 76.0 | 1.12–1.13 | 19.3–19.8 | 11.0–11.8 | 18.3–21.7 | 13.8–14.3 | Range | |||||||||||||||||||
Females (N = 5) | R-13169 | 54.0 | 54.0 | 1.00 | 13 | 9 | 13.5 | 10.4 | 11 | 14 | 0.370 | 6 | 0 | R-13169 | 1 | 0 | 5 | 1 | 2 | 2 | 0 | 4 | 6 | 20 | 9 | 27 | 19 |
R-11913 | 70.0 | 67.0 | 1.05 | 18.2 | 10.9 | 17.4 | 13.0 | 9 | 12 | 0.358 | 5 | 1 | R-11913 | 0 | 0 | 6 | 1 | 3 | 2 | 1 | 3 | 3 | 16 | 9 | 24 | 20 | |
R-13011-2 | 60.0 | 60.0 | 1.00 | 16.6 | 9.6 | 14.8 | 10.7 | 8 | 12 | 0.397 | 4 | 0 | R-13011-2 | 0 | 0 | 5 | 0 | 2.5 | 1 | 1 | 3 | 7 | 17 | 8 | 25 | 18 | |
R-13009-2 | 60.0 | 60.0 | 1.00 | 15.5 | 9.3 | 15.0 | 13.3 | 10 | 15 | 0.333 | 5 | 0 | R-13009-2 | 1 | 0 | 4.5 | 0 | 2.5 | 1 | 1 | 3 | 3 | 18 | 8 | 25 | 18 | |
R-12202 | 54.0 | 51.0 | 1.06 | 13.7 | 8.9 | 13.3 | 11.0 | 8 | 12 | 0.392 | 3 | 0 | R-12202 | 1 | 0 | 5 | 0 | 2.5 | 2 | 1 | 4 | 7 | 18 | 7 | 26 | 18 | |
Range | 54.0–70.0 | 51.0–67.0 | 1.00–1.06 | 13.7–18.2 | 8.9–10.9 | 13.3–17.4 | 10.7–13.3 | 6–11 | 10–15 | 0.33-0.39 | 3–6 | - | Range | - | - | 4.5–6 | – | 1–3 | 1–2.5 | 0–2 | 2–4 | 3–7 | 16–20 | 7–11 | 24–27 | 18–21 | |
Mean ± S.D. | 59.6±6.5 | 58.4±6.2 | 1.02±0.03 | 16.0±1.9 | 9.7±0.9 | 15.1±1.7 | 12.0±1.3 | 9.0±1.8 | 12.6±1.6 | 0.4±0.0 | 4.7±1.1 | - | Mean ± S.D. | - | - | 5.2±0.4 | - | 2.0±0.8 | 1.6±0.5 | 1.0±0.6 | 3.1±0.7 | 5.4±1.8 | 18.1±1.5 | 8.7±1.3 | 25.7±1.3 | 18.9±1.2 |
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.
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 (
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
There is no morphological or mtDNA evidence for recognizing Ph. m. galli as a distinct subspecies; we therefore confirm the conclusions of
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 (
(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 (
Iran, Khorasan historical area, Khorasan Razavi Province (ostan), environs of Gonabad, the right bank of the Kale-Shur River; sand dunes (see Fig.
Ph. mystaceus in life: A subadult Ph. mystaceus khorasanus ssp. n., orange lower surface of the tail is shown, Iran (photograph by R. A. Nazarov) B Ph. mystaceus khorasanus ssp. n., female, Iran (photo by R. A. Nazarov) C Ph. m. mystaceus, Russia, Astrakhan region, Dosang (photograph by E. A. Dunayev) D Ph. m. mystaceus, Dagestan, Sarykum sands (photograph by E. A. Dunayev) E Ph. m. mystaceus, Uzbekistan, Qarakalpaqiston (corresponds to the previously recognized subspecies “galli”; photograph by E. A. Dunayev) F Ph. m. mystaceus, Dagestan, Sarykum sands (corresponds to the previously recognized subspecies “dagestanica”; photograph by E. A. Dunayev) G Ph. m. aurantiacocaudatus, E Kazakhstan, SE Balkash Lake (photograph by E. N. Solovyeva) H Ph. m. aurantiacocaudatus, E Kazakhstan, SE Balkash lake (photograph by E. N. Solovyeva) I Ph. m. mystaceus, Russia, Astrakhan region, Dosang (photograph by E. A. Dunayev).
A member of Ph. mystaceus species complex based on the following combination of morphological attributes: (1) a large-sized Phrynocephalus species with 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 (
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.
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
Head large, rounded, distinctly wider than neck region (see Fig.
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.
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 well-distinct (Fig.
Variations of morphological characteristics in the type series are shown in Table
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.
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
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.
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 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 (SLIV 25.7 (24–27; N = 7) in vs. 30.2 (25–35; N = 70) in Ph. m. mystaceus sensu lato); comparatively lower number of supralabials (SL 12.1 (10–14; N = 7) vs. 14.9 (10-19; N = 70) in Ph. m. mystaceus sensu lato) and by the comparatively shorter black distal part on the tail ventral surface (TL-black/TL 0.38 (0.36–0.40; N = 7) vs. 0.42 (0.32–0.48; N = 70) in Ph. m. mystaceus sensu lato). In life, juvenile and young specimens of the new subspecies can be further distinguished from Middle Asian / Caspian Basin populations of Ph. mystaceus by is rusty orange color of the proximal part of tail ventral surface (vs. lemon-yellow in Ph. m. mystaceus sensu stricto), but is similar to orange tail coloration in juveniles of East Kazakhstan – western China populations described as Ph. m. aurantiacocaudatus.
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 (
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 psammophilous biology of this species, as it was suggested by previous researchers (
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.
On the contrary, the Khorasan population described herein as Ph. m. khorasanus shows very deep genetic divergence in mtDNA which is comparable to the species-level 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
Field studies were supported by the Russian Foundation for Basic Research (grants Nos.: RFBR 16-34-00295-mol-a, RFBR 15-04-08393), morphological and molecular research – by the Russian Science Foundation (RSF № 14-50-00029), sample deposition was conducted within the State Project (AAAA-A16-116021660077-3). Authors are grateful to the colleagues who took part in the fieldwork, material collection and discussion of results: Leonid A. Neymark, Dmitriy A. Bondarenko, Vladimir S. Lebedev, Anna A. Bannikova and Alexander Y. Presnyakov. For permission to study specimens under her care and support, we thank Valentina F. Orlova (
Material examined in morphological analysis.
Phrynocephalus mystaceus mystaceus: Kalmykia (
Phrynocephalus mystaceus galli: Turkmenistan, Repetek (
Phrynocephalus mystaceus aurantiacocaudatus: Kazakystan, Muyunkum sands (
Phrynocephalus mystaceus khorasanus ssp. n.: Iran, Khorasan (
Mann-Whitney test of independent series: “aura” – Ph. m. “aurantiacocaudatus”, “ir” – 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 | ir-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* |
2. TL | 0.044* | 0.000* | 0.001* | 0.000* | 0.027* | 0.000* | 0.000* | 0.005* |
3. SVL/TL | 0.008* | 0.000* | 0.115 | 0.001* | 0.006* | 0.000* | 0.001* | 0.591 |
4. SLbA | 0.015* | 0.004* | 0.031* | 0.178 | 0.855 | 0.180 | 0.014* | 0.813 |
5. SL | 0.007* | 0.001* | 0.050* | 0.003* | 0.455 | 0.005* | 0.003* | 0.355 |
6. TL-black/TL | 0.023* | 0.000* | 0.001* | 0.002* | 0.016* | 0.499 | 0.001* | 0.527 |
7. SSbNb | 0.351 | 0.623 | 0.036* | 0.412 | 0.037* | 0.003* | 0.140 | 0.201 |
8. SbN-SpN | 0.596 | 0.597 | 0.158 | 0.050 | 0.000* | 0.118 | 0.570 | 0.169 |
9. SpN | 0.927 | 0.923 | 0.103 | 0.000* | 0.002* | 0.009* | 0.457 | 0.022* |
10. hSpN SbN | 0.015* | 0.000* | 0.033* | 0.127 | 0.758 | 0.096 | 0.001* | 0.259 |
11. SbN-L | 0.010* | 0.589 | 0.097 | 0.000* | 0.992 | 0.016* | 0.144 | 0.234 |
12. WS&BL | 0.111 | 0.000* | 0.027* | 0.026 | 0.026 | NA | 0.002* | 0.524 |
13. aIMd-IL | 0.017* | 0.392 | 0.053 | 0.113 | 0.771 | 0.252 | 0.030* | 0.091 |
14. SuSSCF | 0.029 | 0.004* | 0.912 | 0.164 | 0.000* | 0.000* | 0.174 | 0.839 |
15. pSL-CF | 0.272 | 0.026* | 0.004* | 0.042* | 0.000* | 0.025* | 0.007* | 0.857 |
16. ILbA | 0.006* | 0.046* | 0.002* | 0.022* | 0.510 | 0.002* | 0.003* | 0.640 |
17. IL | 0.023* | 0.017* | 0.059 | 0.645 | 0.013* | 0.059 | 0.022* | 0.588 |
18. SLIII | 0.009* | 0.011* | 0.008* | 0.011* | 0.738 | 0.002* | 0.001* | NA |
19. FrIII | 0.341 | 0.000* | 0.056 | 0.000* | 0.000* | 0.889 | 0.283 | |
20. SLIV | 0.000* | 0.087 | 0.000* | 0.087 | 0.463 | 0.026* | 0.000* | |
21. FrIV | 0.001* | 0.000* | 0.937 | 0.000* | 0.000* | 0.026* | 0.563 |
MALES | FEMALES | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
aura-ir | ir-myst | ir-galli | myst-aura | aura-galli | galli-myst | ir-all | aura-ir | ir-myst | ir-galli | myst-aura | aura-galli | galli-myst | ir-all | |
1. SVL | 0.432 | 0.142 | 1.000 | 0.004* | 0.533 | 0.002* | 0.110 | 0.147 | 0.001* | 0.003* | 0.000* | 0.007* | 0.001* | 0.003* |
2. TL | 0.694 | 0.034* | 0.160 | 0.002* | 0.328 | 0.000* | 0.726 | 0.015* | 0.001* | 0.003* | 0.000* | 0.052 | 0.000* | 0.001* |
3. SVL/TL | 0.260 | 0.033* | 0.040* | 0.047* | 0.947 | 0.009* | 0.022* | 0.005* | 0.002* | 0.921 | 0.006* | 0.000* | 0.000* | 0.045* |
4. SLbA | 0.279 | 0.028* | 0.550 | 0.057 | 0.215 | 0.003* | 0.281 | 0.039* | 0.086 | 0.028* | 0.861 | 0.547 | 0.837 | 0.040* |
5. SL | 0.043* | 0.032* | 0.293 | 0.037* | 0.063 | 0.005* | 0.043* | 0.048* | 0.009* | 0.045* | 0.038* | 0.771 | 0.101 | 0.023* |
6. TL-black/TL | 0.091 | 0.033* | 0.040* | 0.395 | 0.141 | 0.186 | 0.032* | 0.186 | 0.004* | 0.048* | 0.002* | 0.263 | 0.077 | 0.018* |
7. SSbNb | 0.715 | 1.000 | 0.197 | 0.450 | 0.089 | 0.010* | 0.537 | 0.457 | 0.601 | 0.106 | 0.752 | 0.148 | 0.073 | 0.230 |
8. SbN-SpN | 0.330 | 0.892 | 0.593 | 0.178 | 0.017* | 0.296 | 0.892 | 0.804 | 0.526 | 0.125 | 0.150 | 0.007* | 0.229 | 0.453 |
9. SpN | 0.436 | 0.361 | 0.091 | 0.663 | 0.040* | 0.143 | 0.192 | 0.901 | 0.844 | 0.109 | 0.932 | 0.034 | 0.037* | 0.928 |
10. hSpN SbN | 0.103 | 0.049 | 0.176 | 0.441 | 0.813 | 0.392 | 0.053 | 0.063 | 0.003* | 0.041* | 0.174 | 0.899 | 0.156 | 0.006* |
11. SbN-L | 0.125 | 0.797 | 0.223 | 0.040* | 0.662 | 0.102 | 0.349 | 0.043* | 0.582 | 0.243 | 0.002* | 0.851 | 0.066 | 0.314 |
12. WS&BL | 0.192 | 0.034* | 0.061 | 0.279 | 0.338 | NA | 0.118 | 0.231 | 0.006* | 0.003* | 0.045 | 0.030* | NA | 0.011* |
13. aIMd-IL | 0.528 | 0.454 | 0.294 | 0.520 | 0.466 | 1.000 | 0.611 | 0.002* | 0.130 | 0.007* | 0.097 | 0.818 | 0.208 | 0.004* |
14. SuSSCF | 0.608 | 0.310 | 0.648 | 0.482 | 0.092 | 0.030* | 1.000 | 0.021* | 0.008* | 0.913 | 0.221 | 0.000* | 0.000* | 0.090 |
15 pSL-CF | 0.641 | 0.892 | 0.355 | 0.283 | 0.032* | 0.154 | 0.643 | 0.099 | 0.021* | 0.003* | 0.110 | 0.002* | 0.119 | 0.004* |
16. ILbA | 0.037* | 0.054 | 0.049* | 0.261 | 0.207 | 0.070 | 0.026* | 0.091 | 0.351 | 0.039* | 0.039* | 0.966 | 0.016* | 0.036* |
17. IL | 0.480 | 0.633 | 0.227 | 0.710 | 0.216 | 0.154 | 0.354 | 0.031* | 0.012* | 0.010* | 0.354 | 0.038* | 0.261 | 0.034* |