Acanthosaura aurantiacrista (Squamata: Agamidae), a new long horn lizard from northern Thailand

Abstract Background In Thailand, five species of Acanthosaura have been recorded so far, including Acanthosaura armata from the southern region, A. cardamomensis from the eastern region, A. crucigera from the western region, A. lepidogaster from the northern region and A. phuketensis from the Phuket Island and south-western region. However, comprehensive studies of diversity patterns and distribution of Acanthosaura are still lacking in some areas and need further information for designating areas of special conservation importance and nature protection planning in Thailand. New information Acanthosaura aurantiacrista is a new species of long-horned lizard of the genus Acanthosaura from northern Thailand. It is distinguished from all other species of Acanthosaura by a dagger-like nuchal spine with yellowish-orange colouration in females, bright yellow colouration in males and a combination of other morphological characters: a greater tail length to snout-vent length ratio; a larger postorbital spine, nuchal spine, dorsal spine and occipital spine compared to its head length; a smaller diastema to snout-vent length ratio; a greater number of subdigital lamellae on the fourth finger and fourth toe; and a larger gular pouch than other Acanthosaura species. Analysis of mitochondrial ND2 gene sequences revealed a sister clade between the A. aurantiacrista lineage and the A. crucigera lineage with a 100% probability of divergence, according to Bayesian analysis and strong support value for Maximum Likelihood analysis. The pairwise distance ranged from 13.8-15.0% between A. aurantiacrista and A. cardamomensis, 10.9-14.5% between A. aurantiacrista and A. crucigera and 0-1.2% amongst A. aurantiacrista populations. The discovery of this lizard increases the known endemic herpetological diversity and underscores the importance of conservation in the mountain rainforest region of northern Thailand.

The recent morphological and molecular investigations of Acanthosaura revealed five species across Thailand: Acanthosaura lepidogaster from northern region (Das 2010); A. cardamomensis from the eastern region and Cambodia (Wood Jr. et al. 2010); A. crucigera from the western region and southern Myanmar (Pauwels et al. 2003, Grismer et al. 2008; A. phuketensis from Phuket Island and the south-western region (Pauwels et al. 2015); and A. armata from the southern region (Chan-ard et al. 1999, Pauwels et al. 2002. Hence, one-third of Acanthosaura species have been reported from Thailand so far. A recent field sampling of Acanthosaura species across the Thanon Thong Chai Mountain Range in Thailand revealed three populations: A. crucigera, A. lepidogaster and a new population which has a morphology different from the other species. This population exhibits a large yellowish-orange (females) or bright yellow (males) nuchal crest on the dorsum. To clarify the taxonomic status of this Acanthosaura population, we describe the new species on the basis of its distinctive morphological and colouration characteristics and a genetic analysis. Its name is provided to be included in conservation planning for agamid species in wildlife sanctuaries and national parks in the Thanon Thong Chai Mountain Range.

Specimen collection
Specimens were collected by hand from two locations in Thanon Thong Chai Mountain Range, Thailand: one specimen from Mae Sariang District, Mae Hong Son Province on 27 April 2018; seven specimens from Sop Khong Subdistrict, Omkoi District, Chiang Mai Province on 14 November 2018; and one specimen from Nang Lae Subdistrict, Mueang District, Chiang Rai Province on 24 January 2019. For all specimens, photographs were taken to document their colour patterns prior to euthanisation. Liver samples were taken and stored in absolute ethanol. Specimens were fixed in 10% formalin before being transferred to 70% ethanol for permanent storage. In addition, the comparative morphological characters of other taxa in the genus Acanthosaura were also taken from original descriptions and subsequent studies (Günther 1861, Boulenger 1885, Wood Jr. et al. 2009, Wood Jr. et al. 2010, Ananjeva et al. 2011, Nguyen et al. 2018, Pauwels et al. 2015, Nguyen et al. 2019. For species concepts, the combination of morphological species concept and evolutionary species concept were applied to describe genealogical relationships (Wiley and Lieberman 2011).

Morphological study
Forty-three meristic and measured characters were noted for each specimen of the type series. Measurements were recorded to the nearest 0.01 mm with a Vernier calliper. Measurements were performed on the left side (Wood Jr. et al. 2009, Wood Jr. et al. 2010, Ananjeva et al. 2011. Meristic characters were counted on the left and right sides, respectively. The list and methodology of measurements and meristic counts followed Pauwels et al. (2015): SVL -snout-vent length, measured from the tip of the snout to the tip of the vent; TaL -tail length, measured from the posterior margin of the vent to the tip of the tail; TBW -tail base width, maximum width at tail base; HL -head length, measured from the posterior edge of the rectis of the jaw to the tip of the snout; HW -head width, maximum head width, the width at the level of the tympanum; HD -maximum head height, measured across the parietal region; SL -snout length, measured from the anterior edge of the orbit to the tip of the snout; ORBIT -orbit diameter, measured from the posterior to the anterior edge of the orbit; EYE -eye diameter, measured from the posterior to the anterior edge of the eye; TD -tympanum diameter, measured horizontally from the anterior to the posterior border of the tympanum; TN -scales absent on tympanum (0) or present (1); PS -postorbital spine length, measured from the base to the tip of the spine; NSLmaximum length of the largest spine in the nuchal crest measured from the base to the tip; DS -maximum length of the largest spine in the dorsal crest measured from the base to the tip; WNC -maximum width of the spines in the nuchal crest, measured at the base; 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; FOREL -forelimb length, measured from axilla to the proximal edge of the palmar region; HINDL -hindlimb length, measured from the groin to the proximal edge of the plantar region; SUPRAL -number of supralabials; INFRAL -number of infralabials; VENT -number of ventral scales counted at the midline from the anterior edge of the shoulders to the edge of the vent; FI -number of subdigital lamellae on the fourth finger; TO -subdigital lamellae on the fourth toe; OS -length of the occipital spine, measured from the base to the tip; NSSOS -number of scales surrounding the occipital spine; ; CSnumber of canthus rostralis-supraciliary scales, counted from the nasal scale to the posterior end of the ridge at the posterior margin of the orbit; RW -rostral width; RHrostral height; RS -number of scales bordering the rostral scale; NS -number of scales between the nasals; NCS -number of scales between the fifth canthals; NSCSL -number of scales from the fifth canthal to the fifth supralabial; NR -number of scales between the nasal and the rostral scales; NSSLC -number of scales between the seventh supralabial and the sixth canthal; MW -mental width; MH -mental height; PM -number of scales bordering the mental; YAS -presence (1) or absence (0) of a Y-shaped arrangement of enlarged scales on the snout; ND -presence (1) or absence (0) of a black, diamond shaped, nuchal collar; LKP -presence (1) or absence (0) of a light knee patch; BEPpresence (1) or absence (0) of a black eye patch; ESBO -presence (1) or absence (0) of elliptical scales below the orbit; GP -size of gular pouch scored as absent, small, medium or large; OF -presence (1) or absence (0) of oblique fold anterior to the forelimb insertion.

Molecular techniques
Molecular data were generated for seven specimens from Thanon Thong Chai Mountain Range, with other Acanthosaura taxa and outgroup ( Calotes emma) obtained from GenBank (Macey et al. 1997a, Macey et al. 2000, Zug et al. 2006, Okajima and Kumazawa 2010, Wood Jr. et al. 2010, Yu et al. 2015 (Table 1). Liver tissues were used for DNA extraction with TIANamp Genomic DNA Kit. The samples were lysed using proteinase K for 3-4 hours at 56ºC. The DNA was eluted from the spin column with 200 µl of buffer. Polymerase chain reaction (PCR) was prepared using an EP0402 TAQ DNA POLYMERASE. Two primers, METF6 (L4437a; 5'-AAGCTTTCGGGCCCATACC-3') and ACANTHND2.833. R1 (5'-AGGGAGGTTATTGTTGCTAG-3'), were used to amplify a 698 bp fragment of the NADH dehydrogenase subunit 2 (ND2) gene (Macey et al. 1997b, Kalyabiana-Hauf et al. 2004, Gamble et al. 2008, Wood Jr. et al. 2010. PCR protocol for the amplification of genomic DNA began with an initial denaturation for 2 min at 95ºC, followed by 95ºC for 35 s, annealing at 50ºC for 35 s and extension at 72ºC for 154 s per cycle for 32 cycles ).
Diagnosis: Acanthosaura aurantiacrista sp. n. is differentiated from all other congeners by this combination of characters: A large size (maximum SVL 130.1 mm for males and 119.3 mm for females) and a single long conical spine above the posterior margin of the eye; a large spine on the occiput between the tympanum and nuchal crest; tympanum naked, large, roundish; large developed gular pouch; scales on flanks randomly intermixed with small keeled and small tubercle scales; large nuchal crest with 8 large dagger-like and pointed spines; narrow diastema with 8-9 scales between the nuchal and vertebral crests; vertebral crest composed of large dagger-like, pointed spines beginning at the shoulder region and decreasing in size until the base of the tail; nuchal and dorsal crests are orange in females and yellow in males; tail 1.40-1.70 times the SVL; and black collar and black eye patch present, extending posteriorly until reaching the nuchal crest.

Description
Description of the holotype: Adult female. SVL 105.7 mm; TaL 151.8 mm (1.44 times SVL), tail complete; HL (23.3 mm) slightly longer than HW (18.9 mm); HL one-fifth SVL (0.22 times SVL), HW narrow (0.179 times SVL) and HD tall (0.64 times HL); head triangular in dorsal and lateral views; SL moderately long (0.46 times HL); RW wide (2.31 times RH); steeply sloping anteriorly; CS prominent, forming a large projecting shelf extending above eye, composed of 14/13 large scales; shelf terminates with a notch anterior to postorbital spine; rostrum moderate in size, rectangular, bordered laterally by first SUPRALs and posteriorly by five smaller scales; nostrils roundish, surrounded by one prenasal anteriorly, four postnasals posteriorly and two subnasals; six NS; oval supranasals; large scales above orbit weakly keeled; three rows of moderately-keeled scales below orbit extending from the anterior margin of the eye to posterior; large EYE (0.28 times HL) and ORBIT (0.44 times HL); interorbital, prefrontal and frontal scales slightly keeled and smaller than scales below orbit; seven large, keeled, azygous prefrontal scales arranged in a Y-shaped pattern; parietal eyespot surrounded by a larger row of scales; large conical PS above posterior margin of the eye surrounded by five small lanceolate scales; single row of seven large keeled scales extending from suborbital below posterior margin of eye to above tympanic margin, increasing in size posteriorly; elongated conical OS on lateral margin of nape surrounded by a rosette of five small lanceolate NSSOS; tympanum exposed, roundish, with a size two-thirds that of EYE (0.69 times EYE), surrounded by tiny conical scales; thirteen rectangular SUPRALs similar in size; mental pentagonal, larger than adjacent INFRALs; two postmentals similar in size, four scales contacting PM; chin shields large, extending posteriorly to angle of jaw, separated from infralabials by one scale row anteriorly and three at angle of jaw; eleven rectangular INFRALs of similar size; gular scales sharply keeled and spinose with a larger midventral row; extensible dewlap present; nuchal crest composed of eight elongated, dagger-like scales, bordered on each side by two rows of large, flat, keeled, triangular scales; nuchal crest followed by a diastema of nine DIASN at base of nape; dorsal body crest extending from posterior margin of diastema to base of tail; dorsal crest composed of small laterally compressed, triangular epidermal scales, bordered by a row of smaller paravertebral triangular scales; DS slightly decreasing to sacrum, then fading progressively; and nuchal and dorsal crests present as orange in live specimen (Fig.  2).
Body robust, triangular in cross-section; dorsal body scales small, mixed with some large-keeled scales without a regular pattern, keels projecting posteriorly; pectoral and abdominal scales larger than DS; keeled, semi-transverse rows arranged; keeled scales anterior to large vent; limbs relatively long, dorsal forelimb and hindlimb scales keeled and larger than VENT; five digits on manus; subdigital scales keeled, FI 23/21; five digits on pes; subdigital scales keeled, TO 27/26; TaL 1.44 times SVL, tail covered with keeled spinose scales, keels on subcaudals directed posteriorly; subcaudals much longer than supracaudals; TBW 10.3 mm; and four white creamy eggs with a diameter of approximately 10 mm inside the body.

Variation:
The female paratypes resemble the holotype in all aspects, and the observed differences were a larger PS (7-10.5 vs. 5.7) and OS (5.3-7.5 vs. 4.5) and greater NSL (9.6-12.7 vs. 8.3), wider WNC (0.9-1.6 vs. 0.6), more SUPRAL (   white with some dark spots or dirty brown colouration on the abdomen with creamy brown colouration on pectoral and forelimbs; forelimbs and hindlimbs dark brown dorsally; tail banded with dark brown and dirty light brown (Fig. 4).
In addition, the hemipenis of QSMI1448 is everted approximately 10 mm from the cloaca opening to the hemipenis tip on each side. The hemipenis on each side diverged to a symmetrical spongy millet shape with a width of approximately 5 mm. In preserved ethanol, the hemipenes exhibited a creamy yellow colouration.
Females -Front of head whitish-yellow or creamy white; lips orange-yellow; black eye patch; lateral head and neck yellow intermixed with orange; gular pouch white or yellow intermixed with white; postorbital and occipital spines creamy yellow; nuchal crest yellowish-orange or reddish-orange; dorsal crest reddish-orange; body with brownishgrey or rusty-grey marbled reticulum with some grey keeled scales; whitish or whitish- yellow ocellated spot at the knee and elbow, with certain others indicated on the forelimbs and hindlimbs; ventral creamy white or creamy yellow intermixed with some dark brown on the abdomen, ventral region of forelimbs and hindlimbs; forelimbs and hindlimbs brownish-grey dorsally; tail banded with dark brown and dirty light brown.

Etymology:
The specific epithet aurantiacrista came from a combination of the Latin words aurantiaco (orange) and crista (crest). The name refers to a distinctive characteristic of the first discovered female specimen, which exhibited nuchal and dorsal crests with an orange colour. We suggest the following common names: kingkakhaownaam seesom (Thai), orange crested horned lizard (English), orangeverzierter gehörnter Nackenstachler (German) and Acanthosaurus à crête orange (French).

Ecology
Acanthosaura aurantiacrista sp. n. has been found in evergreen forests on hills up to at least 600 m elevation (Fig. 6). It is active during the day on the ground, logs or rocks or 1-2 m above the ground on trees. During night, it is inactive and sleeps on twigs or trees 1-2 m above the ground. This species displays a defence mechanism against approach or provocation consisting of falling to the ground and running away to find refuge under rocks, log hollows or shrubs.
Sanctuary; Lum Nam Pai Wildlife Sanctuary; Mae Lao-Mae Sae Wildlife Sanctuary; Mae Tuen Wildlife Sanctuary; and Om Koi Wildlife Sanctuary. Moreover, our research team recommends this new long-horned species, whose nuchal and dorsal crests have an appearance similar to fire, as a representative animal for drawing the attention of the local people, scientific community and government towards addressing the current problem of forest burning in northern regions of Thailand.