A new species of the genus Acanthosaura from Yunnan, China (Squamata, Agamidae)

Abstract A new species of Acanthosaura from Yunnan, China is described based on unique morphometric and meristic external characters and a very distinctive color pattern. The fourteenth species recorded of this genus, Acanthosaura tongbiguanensissp. nov., was previously considered A. lepidogaster although it more closely resembles A. crucigera. It can be separated from all other species of the genus by having different numbers of subdigital lamellae on the fourth finger and toe, and a different shape of the black eye patch. The new species differs genetically from investigated congeners by percentage distance of 14.46% to 23.27% (cytochrome b gene).

During our field research in Dehong Autonomous Prefecture, Yunnan Province, China, we discovered some lizards that looked superficially like Acanthosaura lepidogaster. According to Zhao et al. (1999) and Yang et al. (2008), two species of the genus Acanthosaura are distributed in China and only A. lepidogaster is found in Yunnan Province. Morphological and molecular data show that this population is clearly distinct from all other named species, and we consequently describe and name it herein.

Materials and methods
Specimens were collected by hand. Photographs were taken to document color pattern in life prior to euthanasia. Liver tissues were stored in 99% ethanol and lizards were preserved in 75% ethanol. Specimens were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences.
Forty-nine meristic and mensural characters were noted for each adult specimen of the type series, but only meristic characters were noted on juvenile specimens (see Table 3). Measurements were taken to the nearest 0.1 mm with a digital caliper. Paired measurements were made on the left side, as was done in the recent revisions in the Acanthosaura crucigera species group (Wood et al. 2009(Wood et al. , 2010Ananjeva et al. 2011). Paired meristic characters are given as left/right. The list and methodology of measurements and meristic counts follow Wood et al. (2010) and Pauwels et al. (2015):

BEP
presence (1) or absence (0) of a black eye patch; CS number of canthus rostralis-supraciliary scales, counted from the nasal scale to the posterior end of the ridge at the posterior margin of the orbit; DIAS length of the diastema, measured from the posterior end of the nuchal crest to the anterior end of the dorsal crest; DIASN number of scales in the vertebral crest scale diastema, counted from the posterior end of the nuchal crest to the anterior end of the dorsal crest; DS maximum length of the largest spine in the dorsal crest, measured from the base to the tip; DSL longest dorsal scale, measured at the base below the dorsal crest; ESBO presence (1) or absence (0) of elliptical scales below the orbit; EYE eye diameter, measured from the posterior to the anterior edge of the eye; FI number of subdigital lamellae on the fourth finger; FOREL forelimb length, measured from axilla to the proximal edge of the palmar region; GP size of gular pouch, scored as absent (0), small (1), medium (2), large (3) or very large (4); HD maximum head height, measured across the parietal region; HINDL hindlimb length, measured from groin to the proximal edge of the plantar region; HL head length, measured from posterior edge of the lower jaw to the tip of the snout; HW head width, maximum head width, the width at the level of the tympanum; INFRAL number of infralabials; LKP presence (1) or absence (0)  We compared the characters of the new collection with the characters of all currently recognized species of Acanthosaura (Pauwels et al. 2015;Nguyen et al. 2018Nguyen et al. , 2019, see Table 4.
The character DIAS of Acanthosaura brachypoda is given both as 4.5 and 1.9 mm in the original description, which is based on a single specimen, so this character is not used here for comparisons. The methodology for taking FOREL and HINDL was insufficiently described in the original description of A. brachypoda and thus could not be compared here; CS, NCS, NR, NSCSL, NSSLC and NSSOS were not provided in the original description of A. brachypoda; NSSLC, PM, ND, LKP, ESBO and OF were not provided in the original description of A. murphyi; SL, ORBIT, WNC, FOREL, HINDL, VENT, OS, NSSOS, CS, RS, NS, NSC, NSCSL,NR, NSSLC, PM, YAS, BEP, ESBO and GP were not provided in the original description of A. phongdienensis.
Molecular data were generated for three specimens and all available homologous sequences obtained from GenBank, all new sequences have been deposited in GenBank. According to Ananjeva et al. (2008), the sequences whose GenBank accession numbers are AY572873 to AY572886 belong to Acanthosaura nataliae, and the sequences whose GenBank accession numbers are AY572896 to AY572899 belong to Acanthosaura coronata. According to Nguyen et al. (2019), it can be inferred that the sequences whose GenBank accession numbers are AY572900, AY572904, AY572905, AY572912 to AY572918, AY572922 and AY572923 probably belong to Acanthosaura phongdienensis. According to Pauwels et al. (2015), it can be inferred that the sequences whose GenBank accession numbers are AY572887 and AY572889 to AY572894 probably belong to Acanthosaura phuketensis. According to Kalyabina-Hauf et al. (2004) and Ananjeva et al. (2008), the sequences whose GenBank accession numbers are AY572928 to AY572930 belong to some unknown species. Two agamids Pseudocalotes brevipes (Werner, 1904) and Calotes versicolor (Daudin, 1802) were used as the outgroups based on the results from Kalyabina-Hauf et al. (2004). All the GenBank accession numbers for taxa used in the genetic analysis can be found in Table 1. Total genomic DNA was extracted from liver tissue stored in 99% ethanol. Tissue samples were digested using proteinase K, and subsequently purified following a standard phenol/chloroform isolation and ethanol precipitation (Sambrook et al. 1989). PCR was performed using primers new to this paper TBG-F: ATTCTCGCAATACACTACACAAC and TBG-R: TTTCAAATAATACTTGGGAGGTT. Amplification conditions were as follows: after an initial denaturation at 94 °C for 300 s, 31 cycles followed with a denaturation at 94 °C for 45 s, annealing at 42-45 °C for 45 s, and extension at 70 °C for 120 s; cycle sequencing reactions used a two-step program: 15 cycles followed with denaturation at  (Hanke et al. 1994). Amplified mitochondrial cytochrome b (cytb) fragments were sequenced in both directions using an ABI PRISM 3730 Automated DNA Sequencer (Applied Biosystems) following the manufacturer's protocol (Nguyen et al. 2019). Sequences were aligned using CLUSTAL X v1.83 (Thompson et al. 1997) with the default parameters and the alignment revised by eye. Pairwise distances between species were calculated in MEGA 7 (Tamura et al. 2011). The best substitution model HKY+G+I was selected using the Akaike Information Criterion (AIC) in MODEL-TEST v3.7 (Posada and Crandall 1998). Bayesian phylogenetic inference was performed in MRBAYES 3.2.6 (Wang et al. 2009, Ronquist et al. 2012) based on the selected substitution model. Two runs were performed simultaneously with four Markov chains starting from random tree. The chains were run for 1,000,000 generations and sampled every 100 generations. The first 25% of the sampled trees was discarded as burn-in after the standard deviation of split frequencies of the two runs was less than a value of 0.01, and then the remaining trees were used to create a 50% majority-rule consensus tree and to estimate Bayesian posterior probabilities (BPPs). Maximum likelihood analysis was performed in MEGA 7 (Tamura et al. 2011), nodal support was estimated by 1,000 rapid bootstrap replicates.

Results
The obtained sequence alignment is 795 bp in length. Both Bayesian inference and Maximum likelihood analyses recovered this lineage of the new samples as the sister to the clade consisting of Acanthosaura crucigera and A. cf. phuketensis with weak support (Figures 1, 2). The average uncorrected pairwise distances (p-distance) between other investigated members of Acanthosaura ranged from 11.17% to 23.9%, the average Etymology. The name refers to Tongbiguan Nature Reserve, the locality where the new species was found.
Diagnosis. A medium-sized (maximum SVL 115.6 mm) agamid lizard with two pairs of spines: postorbital (supraciliary) spines and spines on occiput between tympanum and nuchal crest; tympanum naked; moderately developed gular pouch; scales on flanks randomly intermixed with medium and large scales; nuchal crest present and strongly developed; diastema between the nuchal and dorsal crests present; dorsal crest slightly developed, composed of enlarged, pointed scales beginning at shoulder region and decreasing regularly in size; tail 1.56-1.85 times SVL; black nuchal collar present; black eye patch present; black oblique folds anterior to the fore limb insertions present.
The new species can be separated from all congeners by having different numbers of subdigital lamellae on the fourth finger (19-21) and toe (25-28), and a different shape of the black eye patch, that extends from posterior margin of nostrils through orbit posteriorly and downwards beyond the posterior end of the tympanum but neither meeting the diamond shaped black nuchal collar on nape nor black oblique humeral fold.
Description of the holotype. Adult male. SVL 110.8 mm. TL 205.0 mm, tail complete. Head length 31.1 mm; head moderately long (HL/SVL 28%), somewhat narrow (HW/SVL 18%), not tall (HD/HL 52%), triangular in dorsal and lateral profile. Snout short (SL/HL 31%); interorbital and frontal regions and rostrum wide,  steeply sloping anteriorly. Canthus rostralis prominent, forming a large projecting ridge extending above eye, composed of 11/13 enlarged scales; the ridge terminates with a notch anterior to the postorbital spine. Rostral moderate in size, rectangular; nasal concave, nostrils surrounded by a circular scale. Eye relatively large (EYE/HL 22%), orbit very large (ORBIT/HL 35%). Prefrontal and frontal scales slightly keeled and larger than scales between supralabials; scales on occiput weakly keeled. Moderately elongate epidermal spine above posterior margin of eye, straight, surrounded by 5/4 enlarged scales. A notch present between the supraciliary edge and postorbital spine. Moderately elongate epidermal spine on occipital region, straight, surrounded by a rosette of 5/4 short spiny scales. Tympanum exposed, oblong, surrounded by small scales. Supralabials 13/13, rectangular, scales in center of series largest; mental squarish above, becoming triangular below, larger than first pair of INFRAL; five scales contacting the mental; infralabials 13/12, rectangular, scales in center of series largest; gulars sharply keeled and spinose. Dewlap extensible, gular pouch moderate. Nuchal crest composed of four very elongate, lanceolate, laterally compressed scales and one moderately elongate, lanceolate, laterally compressed scale bordered on each side by one row of enlarged, spinose scales; nuchal crest followed by a diastema at base of nape. Dorsal body crest slightly developed, extending from posterior margin of diastema onto base of tail; vertebral crest composed of enlarged, epidermal, laterally compressed, spinose scales, bordered by a single row of smaller paravertebral spinose scales; vertebral crest tapers slightly to base of tail, then fades progressively. Body slightly short, triangular in cross-section. Dorsal scales small, mixed with large scales indistinctly arranged in slanted forward and downward rows from the midline of the back, keels projecting posterior wards; scales of pectoral region and abdomen larger than dorsal scales, keeled, more or less arranged in transverse rows; keeled scales anterior to vent not enlarged. Limbs relatively long (FOREL/SVL 39%, HINDL/SVL 56%); dorsal and ventral scales of forelimbs keeled, spinose, about the same size. Five digits on manus; subdigital scales keeled, subdigital lamellae under fourth finger 20/21. Scales of hind limbs keeled and spinose; postfemoral scales small, interspersed with larger spinose scales. Five digits on pes; subdigital scales keeled, subdigital lamellae under fourth toe 26/27. Tail length 1.85 times SVL, tail covered with keeled spinose scales, keels on subcaudals directed posteriorly; subcaudals much longer than supracaudals; base of tail 13.1 mm wide.
Color of holotype in life. Dorsal surface of head black, dorsal surface of body and limbs orangish brown; black eye patch extending from posterior margin of nostrils through orbit posteriorly and downwards beyond the posterior end of the tympanum but neither meeting the diamond shaped black nuchal collar on nape nor black oblique folds anterior to the fore limb insertions; upper lip white, same as color of lateral and ventral sides of neck, lower lip white with small black speckle at posterior region; iris orangish brown; black nuchal collar extending downward to reach black oblique folds anterior to fore limb insertions, two white patches at lower back of black nuchal collar; gular region white; postorbital spines, occipital spines, nuchal crest spines and ridge of the rostral and orbit cream-colored; tongue and inside of mouth pink; few small black speckles and yellow diagonal stripes from midline of the back, irregular light colored spots on sides of body not obvious; stripes checkered with black and white on dorsal ground of limbs and tail; ventral sides of limbs and body white, front part white and back part dark on ventral side of tail. However, it should be noted that this species can change the color of its body within a certain range like most other members of the genus.
Variations. Morphometric and meristic data for the type series are provided in Table 3. The paratypes resemble the holotype in most aspects except that the male KIZL201801 has a darker dorsal ground of the body with no black speckles in the dorsal pattern, and the number of nuchal crest scales is six. The female KIZL201805 has a much darker dorsal ground of the body and irregular black patterns on the ventral sides of the body, limbs, and tail; light colored spots on the sides of the body are more obvious, the color in the gular region is a bit darker. The juveniles KIZL201802 and KIZL201803 have much shorter postorbital, occipital, and nuchal crest spines, and obvious radial patterns around the eyes; the colors of the bodies are relatively darker, the yellow diagonal stripes from the midline of the back are more obvious; they also have irregular black patterns on the ventral sides of the body, limbs, and tail; nuchal crest scales of KIZL201803 numbers four. The females 74II0039 and 74I0040 were not observed alive but only in preservative: the female 74II0039 has a much more obvious black speckling in the dorsal pattern while the female 74II0039 has no black

A. phuketensis; (14)
A. titiwangsaensis. (1) (3) speckles, but they both have irregular black patterns on the ventral sides of the body, limbs, and tail; both have six nuchal crest scales. Distribution. Acanthosaura tongbiguanensis sp. nov. is only recorded in Tongbiguan Nature Reserve including Yingjiang County, Longchuan County and Ruili City, the border region with northern Myanmar in western Yunnan, China, so it probably occurs in northern Myanmar.
Several morphometric characters of Acanthosaura tongbiguanensis sp. nov. overlap with some characters of other species in this genus, however, the new species can be differentiated from all other species of Acanthosaura by the black eye patch extending from the posterior margin of the nostrils through the orbit backwards and downwards to beyond the posterior end of the tympanum but neither meeting black nuchal collar nor the black oblique humeral fold (see Fig. 7).
The Acanthosaura crucigera group is wide ranging and its morphological variation is conserved, it is not surprising to find cryptic diversity within the A. crucigera complex (Wood et al. 2010). Acanthosaura tongbiguanensis sp. nov. was previously considered to represent A. lepidogaster (Yang et al. 2008) although it more closely resembles A. crucigera, however the numbers of subdigital lamellae on the fourth finger and toe of Acanthosaura tongbiguanensis sp. nov. are significantly different from A. lepidogaster and A. crucigera, and the molecular analyses also revealed them distinct taxa. Together with the species described herein Acanthosaura currently comprises 14 species in total.