A new species of the genus Acanthosaura (Squamata, Agamidae) from Yunnan, China, with comments on its conservation status

Abstract A new species of Acanthosaura from Yunnan, China, is described based on morphological and genetic data. The new species can be separated from all other species of the genus by having a different shape of the black eye patch, a different coloration of the postorbital and occipital spines and nuchal crest, and a different color of the gular pouch. Genetically, uncorrected sequence divergences of COI between the new species and investigated congeners ranged from 16.12% to 24.11%. The conservation status of the new species is also discussed.


Introduction
from tissue samples was done by using proteinase K and the standard three-step phenol/chloroform procedure (Sambrook et al. 1989). PCR was performed using primers Chmf4, 5'-TYTCWACWAAYCAYAAAGAYATCGG-3' and Chmr4, 5'-ACYTCRG-GRTGRCCRAARAATCA-3' (Che et al. 2012) in a 25 μl volume with an initial step of 95 °C for 4 min followed by 35 steps of 94 °C for 1 min, 46 °C for 1 min, and 72 °C for 1 min. A final extension involved 72 °C for 10 min. We used a ratio of 0.55 H 2 O : 0.30 ExoI : 0.15 SAP to clean the PCR product (Hanke and Wink 1994). Amplified COI fragments were sequenced in both directions using an ABI PRISM 3730 Automated DNA Sequencer (Applied Biosystems) following the manufacturer's protocol. Sequences were aligned using ClustalW (Thompson et al. 1994) integrated in MEGA 7 (Tamura et al. 2013) with default parameters. Pairwise distances between species were calculated in MEGA 7 (Tamura et al. 2013). The substitution model GTR was selected in MODELTEST v3.7 (Posada and Crandall 1998). Bayesian inference (BI) was performed in MrBayes 3.2.6 (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 (ML) analysis was performed in RaxmlGUI 1.5 (Silvestro and Michalak 2012), and nodal support was estimated by 1,000 rapid bootstrap replicates. Maximum Parsimony (MP) analyses were done using TNT (Goloboff et al. 2008), confidence in tree topology was tested by 1000 generations for non-parametric bootstraps (Felsenstein 1985).
Meristic and mensural characters were noted for each specimen of the type series (Table 3). Measurements were taken to the nearest 0.1 mm with digital calipers. Paired measurements were made on the left side (Wood et al. 2009(Wood et al. , 2010Ananjeva et al. 2011). Paired meristic characters were given as left/right. The methodology of measurements and meristic counts followed Liu et al. (2019):

Results
The obtained sequence alignment is 610 bp in length. BI and ML analyses showed the same result ( Fig. 1) and MP analyses showed a similar result (Fig. 2) to BI and ML. All results indicated that the new species is a separate taxon in the genus Acanthosaura with weak (BI and ML) or strong support (MP). Uncorrected p-distances (Table 2) between investigated congeners ranged from 4.23% to 24.94%. The new species differed from investigated congeners ranging from 16.12% to 24.11% in p-distances.   Paratypes. China • 1 ♀; the same locality as the holotype; 16 May. 2020; Shuo Liu leg.; KIZL2020002 • 2 ♂; the same locality as the holotype; 17 May. 2020; Shuo Liu leg.; KIZL2020003 and KIZL2020004.
Etymology. The specific epithet is dedicated to the renowned herpetologist Prof. Chengzhao Liu (30.9.1900Liu (30.9. -9.4.1976, in recognition of his great contributions to herpetological research in China. Diagnosis. Body size medium (SVL 85.1-95.9 mm), postorbital and occipital spines present; relatively developed gular pouch; scales on flanks randomly intermixed with medium and large scales; nuchal crest gradually developed posteriorly, first nuchal crest spine shortest and last nuchal crest spine longest; diastema between nuchal and dorsal crests present; dorsal crest underdeveloped, composed of enlarged, pointed scales beginning at shoulder region and decreasing regularly in size; tail 1.47-1.77× SVL; number of subdigital lamellae on the fourth finger 16-18 and the fourth toe 22-25; black nuchal collar present; black eye patch extending from nostril through orbit posteriorly and downwards through tympanum and then posteriorly and upwards meeting diamond-shaped black nuchal collar on nape; black oblique folds anterior to forelimb insertions present and not meeting black nuchal collar; anterior nuchal crest spines and dorsal sides of postorbital and occipital spines light colored, posterior nuchal crest spines and ventral sides of postorbital and occipital spines black; gular pouch light blue; tongue and inside of mouth yellow.
Description of the holotype. Adult male. SVL 85.1 mm. TL 150.6 mm, tail complete. Head length 26.7 mm; head moderately long (HL/SVL 31%), somewhat narrow (HW/SVL 22%), not tall (HD/HL 57%), triangular in dorsal and lateral profile. Snout short (SL/HL 40%), a Y-shaped arrangement of enlarged scales on the snout; interorbital and frontal regions and rostrum wide. Canthus rostralis prominent, forming a large projecting shelf extending above eye, composed of 12/13 enlarged scales; shelf terminates with a notch anterior to the postorbital spine. Nasal concave, nostrils surrounded by a circular scale. Eye relatively large (EYE/HL 22%), orbit very large (ORBIT/HL 31%). 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 5/5 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 5/5 short spiny scales. A row of enlarged conical scales present between orbit and occipital spine; a few elongate conical scales present on the posterior of lower jaw. Tympanum exposed, oblong, surrounded by small scales. Supralabials 10/11, rectangular, scales in center of series largest; infralabials 10/11, rectangular, scales in center of series largest; rostral oblong, eight scales contacting the rostral; mental squarish above, becoming triangular below, larger than first pair of infralabials, six scales contacting the mental; gulars sharply keeled and spinose. Dewlap extensible, gular pouch relatively devel- oped. Nuchal crest composed of five elongate, lanceolate, laterally compressed scales bordered on each side by one row of enlarged, spinose scales; nuchal crest gradually developed posteriorly, the first nuchal crest spine shortest and the last nuchal crest spine longest; nuchal crest followed by a diastema at base of nape. Dorsal body crest underdeveloped, extending from posterior margin of diastema onto base of tail; vertebral crest composed of enlarged, epidermal, laterally compressed, spinose scales; vertebral crest tapers slightly to base of tail, then fades progressively. Body slightly short, triangu-  num and then posteriorly and upwards meeting the diamond-shaped black nuchal collar on nape; upper and lower lips white, same as color of lateral and ventral sides of neck; iris reddish brown; black nuchal collar does not reach black oblique folds anterior to fore limb insertions; gular pouch light blue; dorsal sides of postorbital and occipital spines same color as dorsal surface of head, ventral sides of postorbital and occipital spines black, first four nuchal crest spines almost white, last nuchal crest spine black; tongue and inside of mouth yellow; a few almost symmetrical large black and greyish white speckles on both sides of dorsal crest, many small black and light purplish grey speckles on lateral sides of body; dorsal surface of limbs brown with indistinct lightly colored transverse stripes, light patches on elbows and knees; ventral sides of limbs and body white with many small brown spots; brownish black and greyish white rings on tail. However, these lizards can change their body color within limits like most other members of this genus.
Color of holotype in preservative. The coloration in preservative resembles that in life. Light blue on gular region faded; dorsal surface of head and upper and lower lips became darker; spots on belly became indistinct.
Variation. Morphometric and meristic data for the type series are provided in Table 3. The female paratype resembles the holotype in most aspects except that it has a larger body size and a shorter tail, a darker dorsal ground-color of the body, and with its penultimate nuchal crest spine being half black; the light blue color on gular region is indistinct, and the base of the tail is not thickened. The two male paratypes resemble the holotype in most aspects except that they have bigger body sizes and relatively shorter tails, no spot on the ventral sides of their bodies, and both have seven nuchal crest spines.
Distribution. After our extensive field investigation, Acanthosaura liui sp. nov. is only recorded in Jianshui County, Gejiu City, and Shiping County, Honghe Autonomous Prefecture, Yunnan, China.
Acanthosaura liui sp. nov. differs from A. armata, A. aurantiacrista, A. cardamomensis, and A. phuketensis by having much shorter postorbital, occipital, nuchal crest, and dorsal crest spines (nuchal and dorsal spines <10 mm in the new species vs >10 mm in the latter species).
Acanthosaura liui sp. nov. differs from A. bintangensis by the presence of a light knee patch (vs absent in the latter species), the absence of an enlarged row of keeled scales below orbit (vs present), the absence of large yellow spots edged in blackish brown arranged on body and base of tail (vs present); the black eye patch in Acanthosaura liui sp. nov. extends backwards and downwards through the tympanum and continues backwards and upwards to reach the black nuchal collar while it never extends onto the head side in A. bintangensis.
Acanthosaura brachypoda is known only from the single holotype (Ananjeva et al. 2011), so we only compared the female Acanthosaura liui sp. nov. with the characters of the female holotype of A. brachypoda. Acanthosaura liui sp. nov. differs from A. brachypoda by the presence of gular pouch (vs absent). Acanthosaura liui sp. nov. does not have pairs of transverse creamy spots along both sides of spine forming a symmetrical pattern present as in A. brachypoda. The nuchal crest is gradually developed posteriorly, the first nuchal crest spine is shortest, and the last nuchal crest spine is longest, while the nuchal crest is composed of two enlarged terminal (anterior and posterior) scales and much smaller scales between them in A. brachypoda. Acanthosaura liui sp. nov. differs from A. coronata by the presence of obvious postorbital spines, occipital spines, nuchal and dorsal crests (vs absent or not obvious), a diastema between nuchal crest and dorsal crest (vs a continuous nuchal and dorsal crest), the presence of a black nuchal collar (vs absent), the presence of a black eye patch (vs absent), and the presence of a gular pouch (vs absent).
Acanthosaura liui sp. nov. differs from A. crucigera (and the synonymous A. horrescens Lönnberg, 1916) by in that its black eye patch extends backwards and downwards through the tympanum and backwards and upwards to the black nuchal collar, while in A. crucigera the black eye patch only extends to the anterior edge of the tympanum. In the new species the black nuchal collar does not extend downwards to reach the black oblique humeral fold, while in A. crucigera the black nuchal collar extends downwards to reach the black oblique humeral fold.
Acanthosaura lepidogaster is a complex of cryptic species and has a very wide distribution including Vietnam, Thailand, Myanmar, Laos, and southern China, and its type locality is uncertain (Ananjeva et al. 2008(Ananjeva et al. , 2011Nguyen et al. 2019). So, except for the type specimens, other populations of this complex are referred to as A. lepidogaster sensu lato. Thus, we compared Acanthosaura liui sp. nov. with the the original description (Cuvier 1829) and photographs of the type specimen (MNHN 5076) (Ananjeva et al. 2011) of A. lepidogaster sensu stricto. Acanthosaura liui sp. nov. differs from A. lepidogaster in having relatively fewer nuchal crest spines and a relatively larger gular pouch. The nuchal crest is gradually developed posteriorly in the new species; from the second nuchal crest spine, each spine is obviously longer than the previous one, while nuchal crest spines are almost the same length except for the first one in A. lepidogaster. The distance between the two occipital spines are almost equal to the distance between the two postorbital spines in the new species, while the distance between the two occipital spines are obviously less than the distance between the two postorbital spines in A. lepidogaster. Three significantly projecting conical scales above the tympanum make the head appear distinct triangular from above in the new species, while in A. lepidogaster there are some slightly projecting conical scales above the tympanum, but the head is not distinctly triangular from above. For A. braueri Vogt, 1914, A. fruhstorferi Werner, 1904, A. hainanensis Boulenger, 1900, and A. lamnidentata Boulenger, 1885, which have been synonymized with A. lepidogaster, some of them may represent valid species in the A. lepidogaster complex and need re-evaluation. According to the literature (Boulenger 1885(Boulenger , 1900Werner 1904;Vogt 1914;Hallermann 2000), Acanthosaura liui sp. nov. can be distinguished from all of them by the following characters: occipital spine obviously longer than postorbital spine, gular pouch well developed, dorsal surface of head light colored, and black eye patch extending backwards and downwards through the tympanum and then backwards and upwards to reach the black nuchal collar. Acanthosaura liui sp. nov. differs from A. phongdienensis in having a diastema between the nuchal crests and dorsal crests (vs a continuous nuchal and dorsal crest). The black eye patch in Acanthosaura liui sp. nov. extends backwards and downwards through the tympanum and then backwards and upwards to reach the black nuchal collar, while in A. phongdienensis it extends backwards and upwards above the tympanum to reach the black nuchal collar.
Acanthosaura liui sp. nov. differs from A. titiwangsaensis by the presence of light knee patch (vs absent), the absence of medium-sized, light orange spots edged with a faded black color on the body and base of the tail (vs present); the black eye patch in Acanthosaura liui sp. nov. extends backwards and downwards through the tympanum and continues backwards and upwards to reach the black nuchal collar, while in A. titiwangsaensis it is restricted to the orbit and does not extend into the postorbital region.
Acanthosaura liui sp. nov. differs from A. tongbiguanensis in having shorter postorbital and occipital spines. The black eye patch in Acanthosaura liui sp. nov. extends backwards and downwards through the tympanum and continues backwards and upwards to reach the black nuchal collar, while in A. tongbiguanensis it extends backward and downward beyond the posterior end of the tympanum but never continues backward and upward to reach the black nuchal collar. The black nuchal collar does not extend downwards to reach the black oblique humeral fold, while in A. tongbiguanensis it extends downwards to reach the black oblique humeral fold. The tongue and the inside of the mouth are yellow in Acanthosaura liui sp. nov., while they are pink in A. tongbiguanensis. The gular region is light blue in the new species, while it is white in A. tongbiguanensis.

Discussion
The sequence with GenBank accession number MG935416 was identified as Acanthosaura crucigera by Mulcahy et al. (2018); however, the specimen has very long postorbital spines, occipital spines, nuchal crest spines, and dorsal crest spines (Mulcahy et al. 2018: fig. 4), and most similar to A. phuketensis. Additionally, the collection site of this specimen is near Phuket Island, Thailand, the type locality of A. phuketensis. Thus, we consider this specimen as A. cf. phuketensis.
Previously, there were two known species of Acanthosaura in Yunnan, China: A. lepidogaster complex and A. tongbiguanensis. Acanthosaura tongbiguanensis is only found in Dehong Autonomous Prefecture (Liu et al. 2019), western Yunnan, and A. lepidogaster complex occurs in western, southwestern, southern, and southeastern Yunnan (Zhao et al. 1999;Yang and Rao 2008). Acanthosaura liui sp. nov. is only found in south-central Yunnan. To date, no overlap between the distributions of the three species was found.
In areas where Acanthosaura liui sp. nov. occurs, the primordial forest has been severely destroyed and reforested with exotic trees. The forests are now surrounded by farmland and villages, and the habitat is very fragmented. Furthermore, many roads have been built and more roads are being built locally. This is detrimental, as we found that many lizards and other reptiles had died on these roads (Fig. 10). Considering the restricted habitat and distribution of the new species, we suggest International Union for Conservation of Nature status as Endangered. We have suggested to the local forestry department that this endemic species be added to the protected animal lists and that the habitat of this species be protected.