Distribution extension of Calotes irawadi Zug, Brown, Schulte & Vindum, 2006, previously confused with C. versicolor (Daudin, 1802): first record from China

We report the first country record of Calotes irawadi, identified previously as C. versicolor, from China based on four specimens collected from Tongbiguan Nature Reserve, Western Yunnan, China. Morphologically, the specimens show good agreement with the original description of C. irawadi, and phylogenetically clustered with specimens (including holotype) of C. irawadi from Myanmar with strong support. This is also the first record of C. irawadi from outside Myanmar.


Introduction
The agamid genus Calotes Cuvier, 1816 currently consists of 25 species (Uetz et al. 2021). Six of them are recorded in China, namely: C. emma Gray, 1845; C. jerdoni Günther, 1870; C. medogensis Zhao & Li, 1984;C. mystaceus Duméril & Bibron, 1837;C. paulus (Smith, 1935) and C. versicolor (Daudin, 1802) [Wang et al. 2020]. The Garden Fence Crested Lizard C. versicolor, an agamid lizard found commonly across the Indian subcontinent and the Indo-Chinese region, has a complicated taxonomic history because it was described without a locality (Matyot 2004; euthanized with ethyl acetate and then fixed in 75% ethanol for storage after taking photographs. Liver tissue samples were preserved in 99% ethanol for molecular analysis. The specimen was deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ), Yunnan, China.
Total genomic DNA was extracted from liver tissues with a universal protocol of DNA extraction (Aljanabi and Martinez 1997). A region of the mitochondrial gene NADH dehydrogenase subunit 2 (ND2) and its flanking tRNAs was amplified and sequenced by using the primers L3705 (5'-ATT AGG GTC TGC TAC ACA AGC AGT TGG-3') and H5162 (5'-GGT TGA RAG TAR TCA TCG AGT TAA GAA CGAC-3') (Huang et al. 2013). PCR was conducted as in Huang et al. (2013): an initial denaturing step at 95 ℃ for 4 min; 35 cycles of denaturing at 94 ℃ for 35 s, annealing at 65 ℃ for 45 s, and extending at 72 ℃ for 90 s; and a final extending step of 72 ℃ for 8 min. PCR products were electrophoresed in 0.8% agarose gels, visualized with ethidium bromide. The products were purified and sequenced by Invitrogen Trading (Shanghai) Co., Ltd, using the same primers as in PCR. All new sequences were deposited in GenBank. Draco blanfordii Boulenger, 1885; Pseudocalotes kakhienensis (Anderson, 1879) and Gonocephalus grandis (Gray, 1845) were chosen as outgroups based on Zug et al. (2006) and Giri et al. (2019). Homologous and outgroup sequences were obtained from GenBank (Table 1).
Sequences were aligned using ClustalW (Thompson et al. 2002) with default parameters in Mega X (Kumar et al. 2018). The genetic distance (uncorrected p-distance) between species was calculated in Mega X (Kumar et al. 2018) with pairwise deletions of missing data and gaps. The substitution model GTR+G was used according to Giri et al. (2019). Bayesian Inference (BI) was performed in MrBayes v3.2.6 (Ronquist et al. 2012). Two runs were performed simultaneously with four Markov chains starting from the random tree. The chains were run for 1 000 000 generations and sampled every 100 generations. The first 25% of the sampled trees were discarded as burn-in after the standard deviation of split frequencies of the two runs was less than 0.01. The remaining trees were then used to create a consensus tree and to estimate Bayesian posterior probabilities. Maximum Likelihood (ML) analysis was performed in RaxmlGUI 1.5 (Silvestro and Michalak 2012) and nodal support values were estimated by 1,000 rapid bootstrap replicates.
Measurements were taken with a digital caliper to the nearest 0.1 mm, except tail length (TL) which was measured using a string and a ruler. Morphological terminology followed Zug et al. (2006). Morphometric characters included: Eye-ear length (EyeEar, distance from anterior edge of tympanum to posterior of orbit); Head height (HeadH, dorsoventral distance from top of head to underside of jay at transverse plane intersecting angle of jaws); Head length (HeadL, distance from anterior edge of tympanum to tip of snout); Head width (HeadW, distance from left to right outer edge of temporal or jaw muscles at their widest point without compression of soft tissue); Interorbital width (Interorb, transverse distance between anterodorsal corners of left and right orbits); Jaw width (JawW, distance from left to right outer edge of jaw angles: this measurement excludes jaw musculature broadening of head); Naris-eye length (NarEye, distance from anterior edge of orbit to posterior edge of naris); Snout-eye length (SnEye, distance from anterior edge of orbit to tip of snout); Snout width (SnW, transverse distance between left and right nares); 4 th finger (4FingLng, distance from juncture of 3 rd and 4 th digits to distalmost extent of 4 th finger); 4 th toe (4ToeLng, distance from juncture of 3 rd and 4 th digits to distal end of 4 th digit on hindfoot); Crus length (CrusL, length of crus from knee to heel); Forefoot length (ForefL, distance from proximal end of forefoot to tip of fourth digit); Hindfoot length (HindfL, distance from proximal end of hindfoot to distalmost surface of fourth toe); Lower arm length (LoArmL, distance from elbow to distal end of wrist); Pectoral width (PectW, distance between left and right axilla, posterior to forelimb insertions, measured on ventral side); Pelvic width (PelvW, distance between left and right inguen, posterior to

Results
BI and ML analyses shows the same topology, consistent with that of Zug et al. (2006) and Giri et al. (2019). The specimens collected from Tongbiguan NR, Western Yunnan, China, clustered with Calotes irawadi from Myanmar (including holotype); this was strongly supported by both BI and ML (Fig. 1). The genetic distance (uncorrected p-distance) between species within the genus ranged from 5.33% to 38.02% and the genetic distance (uncorrected p-distance) between the specimens from China and C. irawadi from Myanmar (including holotype) was 1.58% (Table 2). Furthermore, morphological characters of the specimens from China agreed with the original description of C. irawadi by Zug et al. (2006). Therefore, we considered that the specimens from Western Yunnan, China belong to C. irawadi.   Table 3. Head is triangular and distinct from neck; snout-tip blunt; head behind eyes with edges slightly bowed outward by jaw muscles but edges largely parallel; sides of head flat; dorsally head scales are variable in size and smooth surfaced, most equivalent in size to dorsal trunk scales; 6-7 scales on line transversally between left and right nasal scales; 8-9 elongate and sharply folded scales along dorsolateral snout ridge from above posterodorsal corner of nasal scale to and including the posterior most supraciliary scale; rostral equivalent to the supralabials in height; supralabials 11; laterally head with single large nasal scale on each side abutting rostral; loreal and preocular area with small scales. The tympanum is large and naked with a pair of spines or clusters in supratympanum area; medially the chin throat scales triangular and smooth to lightly keeled; mental triangular; intralabials 9-10.
Trunk scalation generally keeled dorsally and laterally; middorsal crest of elongate scales; the dorsal spines scales are blade-like and laterally compressed; 44-53 middorsal scales, 38-46 scale rows around trunk at midbody, all trunk scales are keeled, weakly so on ventrolateral half of neck and trunk. Keel and scale orientation are diagonally upward from neck and supra-axillary area to base of tail; preaxillary scales mostly smooth; ventral scales large and uniform in size from throat to vent and strongly keeled.
Limbs have modest to large scales, all keeled; 19-23 lamellae on fourth finger and 23-25 lamellae on fourth toe; each finger and toe with strongly bicarinate lamellae ventrally, whilst claws are long, thin and sharply pointed on all digits.
Tail length is 2.7-3.1 times of SVL; tail scalation similar to trunk although more strongly keeled with progressive loss of scale rows distally.
Coloration. These lizards have a very clear ability to change their body colors. In life, body color varies from yellowish white to almost wholly black with or without dark or light stripes (Fig. 3A, B). In preservative, the dorsal color is dark brown with some indistinct black or white stripes; the ventral color is white with some black stripes (Fig. 2B).
Sexual dimorphism. The width of the base of tail in adult males is significantly thicker than that in adult females, except for this, there is little difference in morphological characters between females and males, and there is little difference in body color between males and females during the nonbreeding season. It is difficult to distinguish the gender of the juveniles in appearance.
Ecological notes. The specimens from Nabang Town and from Hongbenghe were found on the sides of a small road (Fig. 3C) near a village and on the sides of a big road  ( Fig. 3D), respectively. Specimens were collected during both the day and night. They were active on the trunks near the ground during the day, and asleep on the higher branches at night. Reproductive behavior was not observed.

Discussion
In Yunnan Province, China, Calotes versicolor was recorded from Wenshan Prefecture, Dehong Prefecture, Nujiang Prefecture, Baoshan City, Dali Prefecture, Pu'er City, and Xishuangbanna Prefecture (Zhao et al. 1999;Yang and Rao 2008). After many years of field surveys in Yunnan and examinations of related specimens, we have never found C. versicolor in Xishuangbanna and the specimens that were identified as C. versicolor collected from Xishuangbanna are actually C. emma. Therefore, we consider the record of C. versicolor from Xishuangbanna as probably incorrect. Additionally, during surveys, we found that the lizards previously identified as C. versicolor from Eastern Yunnan (Wenshan Prefecture) and Western Yunnan (Dehong Prefecture), belong to two different species. Based on morphological comparisons and molecular analyses, we found that the species previously identified as C. versicolor from Dehong Prefecture is actually C. irawadi. As for the species distributed in Nujiang Prefecture, Baoshan City, Dali Prefecture, and Pu'er City, because we have not collected specimens from these places yet, whether they also belong to C. irawadi remains unknown.
Calotes irawadi was known previously only from the Central Dry Zone in Sagaing, Magwe, Mandalay divisions of Myanmar (Zug et al. 2006). This is the first record of C. irawadi from China and from outside of Myanmar. The new localities in China are approximately 210-240 km away from the type locality in Myanmar (Fig. 4). Our work brings the total species number of the genus Calotes in China to seven.