The Stoor Hobbit of Guangdong: Goniurosaurus gollum sp. nov., a cave-dwelling Leopard Gecko (Squamata, Eublepharidae) from South China

Abstract A new species of the genus Goniurosaurus is described based on three specimens collected from a limestone cave in Huaiji County, Guangdong Province, China. Based on molecular phylogenetic analyses, the new species is nested within the Goniurosaurus yingdeensis species group. However, morphological analyses cannot ascribe it to any known species of that group. It is distinguished from the other three species in the group by a combination of the following characters: scales around midbody 121–128; dorsal tubercle rows at midbody 16–17; presence of 10–11 precloacal pores in males, and absent in females; nuchal loop and body bands immaculate, without black spots; iris orange, gradually darker on both sides. The discovery of yet another limestone-adapted species of Goniurosaurus in Guangdong Province underscores a growing body of evidence for the high biodiversity of limestone habitats and brings into sharp focus the urgent need for their conservation.


Introduction
The eublepharid genus Goniurosaurus Barbour, 1908, currently contains 22 species that are scattered throughout much East and northern Southeast Asia (Uetz et al. 2020;Zhu et al. 2020;Qi et al. 2020). All species of Goniurosaurus have generally restricted, circumscribed ranges and many are restricted to habitats with either granite or limestone (karst) rock (Wang et al. 2014;Liang et al. 2018;Qi et al. 2020). Recent molecular phylogenetic analyses resulted in the partitioning of Goniurosaurus into four monophyletic species groups Qi et al. 2020;Zhu et al. 2020), namely the G. kuroiwae, G. lichtenfelderi, G. luii and, G. yingdeensis groups. The morphological comparisons also sustain the recognition of the G. kuroiwae and G. yingdeensis groups, however, the definitions of the G. lichtenfelderi and G. luii groups still require clarification (Qi et al. 2020).
Currently, three narrowly distributed species within the Goniurosaurus yingdeensis group are known from the karst environments of northern Guangdong, China: G. yingdeensis Wang, Yang & Cui, 2010, G. zhelongi Wang, Jin, Li & Grismer, 2014and G. varius Qi, Grismer, Lyu, Zhang, Li & Wang, 2020. These three species share consistent morphological features that differentiate them from species of the other groups: (1) base of claws sheathed by four scales, two lateral scales of claw short and shell-shaped; (2) precloacal pores fewer than 15 in males and absent in most females (precloacal pores present in females in G. yingdeensis only); precloacal pores form a continuous transverse series not extending onto the femora; (3) enlarged row of supraorbital tubercles indistinct or absent; (4) nuchal loop rounded posteriorly; and (5) four body bands between the nuchal loop and the caudal constriction.
During our herpetological surveys in Guangdong Province, China, three specimens of Goniurosaurus were collected from a new locality during May 2020. Morphological and molecular analyses place this new population within the Goniurosaurus yingdeensis group but cannot ascribe them to any of the known species. We therefore describe this population as a new species.

Sampling
Three specimens of the undescribed species were collected from Huaiji County, Guangdong Province, China. Following euthanasia, all specimens were fixed in 10% formalin and transferred to 75% alcohol. Tissue samples were preserved in 99% alcohol and stored at -40 °C. All specimens are deposited in The Museum of Biology, Sun Yat-sen University (SYS). Sequences of other species of Goniurosaurus generated by Qi et al. (2020) were accessed from GenBank.

Morphological characters
Measurements were taken following Ziegler et al. (2008) using digital calipers (Neiko 01407A Stainless Steel 6-Inch Digital Caliper, USA) to the nearest 0.1 mm. Abbreviations of morphological characters are as follows: SVL snout-vent length (from tip of snout to vent); TaL tail length (from vent to tip of tail); HL head length (from tip of snout to posterior margin of ear opening); HW maximum head width; SE snout-to-eye distance (measured from tip of snout to the boney anterior margin of the orbit); EE eye-to-ear distance (from the boney posterior margin of the orbit to posterior margin of ear opening); SPL supralabials; IFL infralabials; N nasal scales surrounding nare; IN internasals; PostIN granular scales bordering the internasals; PM postmentals; GP gular scales bordering postmentals; CIL eyelid fringe scales or ciliaria; PO preorbital scales (number of scales in a line from posterior margin of external naris to anterior margin of the boney orbit); GST granular scales surrounding dorsal tubercles; PTL paravertebral tubercles between limb insertions; DTR longitudinal dorsal tubercle rows at midbody; MB scales around midbody; PP precloacal pores; PAT postcloacal tubercles. Bilateral scale counts are given as left/right.
Morphological character data of known congeners were taken from the literature (Grismer et al. 1999(Grismer et al. , 2002Orlov et al. 2008;Ziegler et al. 2008;Blair et al. 2009;Wang et al. 2010Wang et al. , 2013Wang et al. , 2014Yang and Chan 2015;Zhou et al. 2018;Qi et al. 2020;Zhu et al. 2020) and from 34 examined museum specimens of the eight species listed in the Appendix.

Phylogenetic analysis
Fifty-one sequences from 13 known Goniurosaurus species plus one outgroup sequence from the eublepharid Hemitheconyx taylori Parker, 1930, which was used to root the tree, were obtained from GenBank and composed the dataset (Table 1). DNA sequences were aligned by the Clustal W with default parameters (Thompson et al. 1997) and trimmed with gaps partially deleted in MEGA 6 (Tamura et al. 2013). Two gene segments, with 486 base pairs (bp) of 16S and 396 bp of Cytb, were concatenated seriatim into an 882 bp sequence, and further divided into two partitions based upon each gene. The partitions were tested in jmodeltest v2.1.2 with Akaike and Bayesian information criteria, all resulting the best-fitting nucleotide substitution models of GTR+I+G. Sequence data were analyzed using Bayesian inference (BI) in  (Ronquist et al. 2012), and maximum likelihood (ML) in RaxmlGUI 1.3 (Silvestro and Michalak 2012). Two independent runs were conducted in the BI analysis with 10,000,000 generations each and sampled every 1000 generations with the first 25% of samples discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of < 0.005. In the ML analysis, a bootstrap consensus tree inferred from 1000 replicates was generated. Nodes with Bayesian posterior probabilities (BPP) ≥0.95 and ML support values of ≥70 were considered strongly supported (Huelsenbeck et al. 2001;Wilcox et al. 2002). Uncorrected pairwise sequence divergences utilizing the 16s gene were calculated using MEGA 6.

Results
The ML and BI analyses resulted in identical topologies ( Fig. 1) and maintained a high degree of consistency with recent molecular phylogenetic analyses Qi et al. 2020). Uncorrected pairwise sequence divergences are reported in Table 2. The phylogenetic analyses support previous analyses indicating that Goniurosaurus can be divided into four strongly supported clades, namely the G. kuroiwae group, G. lichtenfelderi group, G. luii group and the G. yingdeensis group. The Goniurosaurus yingdeensis group is composed of four species with genetic differences among them ranging from 5.5-5.9%. Three of the species are G. varius, G. yingdeensis and G. zhelongi and the fourth species is composed of the individuals of the new population from Guangdong Province. All specimens were recovered as monophyletic with a high nodal support (1.00 in BI and 100 in ML) and low intrapopulational genetic differentiation (0.0-0.5%; Table 2). But due to low nodal support, the phylogenetic relationships among G. varius, G. yingdeensis and G. zhelongi are still unclear. Additionally, the Guangdong population has a combination of morphological characteristics distinguishing it from other species in the G. yingdeensis group as well as showing significant morphological differences from all known congeners. Thus, we hereby describe the specimens from Guangdong Province as a new species.  four scales, dorsal claw scales small, two lateral claw scales short and shell-shaped; (11) axillary pockets deep; (12) presence of 10 or 11 precloacal pores in males and absent in females; (13) dorsal ground color of head, body, and limbs in adults yellowish brown and mottled with irregularly shaped dark-brown blotches; (14) nuchal loop complete, rounded posteriorly; (15) presence of three or four thin dorsal body bands between nuchal loop and caudal constriction, with black anterior and posterior borders, bands usually irregularly shaped; (16) iris orange, gradually darker on both sides. Comparisons. Goniurosaurus gollum sp. nov. can be distinguished from the other 21 known species in the genus by the following combination of characters: base of claws being sheathed by four scales, two lateral claw scales short and shell-shaped (vs. claws sheathed by four scales, two lateral scales of claw long, curved in G. lichtenfelderi group and G. luii group, and not sheathed in G. kuroiwae group); having 10 or 11 precloacal pores in males (vs. 17-46 in G. lichtenfelderi group, 16-33 in G. luii group and absent in G. kuroiwae group); and lacking an enlarged row of supraorbital tubercles (present in G. lichtenfelderi group and G. luii group).

Goniurosaurus gollum
Goniurosaurus gollum sp. nov. can be distinguished from its closest relatives in the Goniurosaurus yingdeensis group by the following combination of characters: scales around midbody 121-128 (vs. 101-110 in G. varius, 101-116 in G. yingdeensis, 99-109 in G. zhelongi); longitudinal dorsal tubercle rows at midbody 16 or 17 (vs. 21-24 in G. varius, 20-25 in G. yingdeensis, 23-28 in G. zhelongi); absence of ten precloacal pores in females (vs. present in G. yingdeensis); nuchal loop and body bands im- maculate (vs. having black spots in G. varius); iris orange, gradually darker on both sides (vs. iris orange-red in G. varius, iris gray and becoming orange near the pupil in G. yingdeensis, iris gray-white and tinged with orange in G. zhelongi). Additional comparisons of morphological characteristics are provided in Table 4 and Figure 4.
Description of holotype. Adult male with original tail; SVL 93.4 mm; HL 24.2 mm; HW 16.2 mm; SE 9.3 mm; EE 9.3 mm; SVL:HL 3.9; HL:HW 1.5; SE:EE 1. Head triangular, wider than neck, covered with granular scales, densely interspersed with tubercles in the temporal and occipital regions; area between orbits uniformly covered by small granular scales; supraorbital tubercles nearly uniform in size; scales of rostrum slightly larger than those in between orbits; rostral convex and hemi-elliptic, 1.8 times as broad as high, middorsal portion of rostral partially sutured dorsomedially, bordered laterally by first supralabial and prenasal, dorsolaterally by supranasal, dorsally by one internasal; external nares oval, surrounded by 8/9 nasals each, anteriorly by prenasal and supranasal, dorsally by supranasal and a granular scale, posteriorly by 7/8 smaller granular scales, and ventrally by the prenasal; prenasal with long recurved ventral portion; supranasals large, separated by one internasals; supralabials rectangular, 10/10; preorbital scales 16/17; eyes relatively large, pupils vertical; eyelid fringe scales 59/60; outer surface of upper eyelid composed of granular scales of about same size of those on top of head; external auditory meatus circular, tympanum deeply recessed; mental triangular, bordered laterally by first infralabial and posteriorly by three postmentals; infralabials rectangular, 10/10; gular scales juxtaposed uniform granular, abruptly into flat juxtaposed pectoral scales, and grading posteriorly imbricated larger ventral scales. Tongue with a small notch at tip. Crowns of teeth expanded, occlusal margins bearing multiple ridges.
Dorsal surface of neck and body covered with uniform granular scales, interspersed with densely sharply pointed conical tubercles; scales around midbody 125; longitudinal rows of dorsal tubercles at midbody 16; vertebral row of scales indistinct; paravertebral tubercles between limb insertions 25; dorsal body tubercles surrounded by 9-10 granular scales; dorsal scales grading ventrally into larger flattened imbricate ventral scales; ten precloacal pores in a transverse series; postcloacal region greatly swollen, covered with imbricate flattened scales, containing 2/2 postcloacal tubercles laterally at level of the vent.
Tail original, long and thin, thickest at base, bearing whorls anteriorly, gradually narrowing to the tip; composed of nine recognizable annuli anteriorly that are 8-9 scales wide, annuli fade abruptly posteriorly into flat juxtaposed scales; incorporating 2-8 sharply pointed conical tubercles in a transverse row, tubercles do not encircle the tail; ventral caudals larger and more nearly square than dorsal caudals.
Limbs relatively long and slender; dorsal surface covered with granular scales, densely interspersed with tubercles; ventral surface covered by flat scales, juxtaposed, subimbricate or imbricate; dorsal surface of pes and manus covered with granular scales, interspersed with several conical tubercles on top of pes, lacking tubercles on top of manus; hind limbs slightly larger than forelimbs; ventral surfaces of pes and manus covered with large granular scales; axillary pockets deep; subdigital lamellae wide, 10/10 on Finger I, 12/14 on Finger II, 15/16 on Finger III, 16/14 on Finger IV, 13/14 on Finger V, 12/12 on Toe I, 15/ 16 on Toe II, 20 / 19 on Toe III, 22 / 23 on Toe IV, and 18 / 20 on Toe V; fingers laterally compressed, relative finger lengths I<V<II<IV<III; toes laterally compressed, third toe nearly as long as the fourth toe, relative toe length I<II<V<III<IV; base of claws sheathed by four scales, two lateral scales of claw short, asymmetrical shell-shaped.
Coloration in life. Dorsal ground color of head, neck, body, and limbs yellowish brown, mottled with irregularly shaped dark-brown blotches; nuchal loop complete and rounded posteriorly, anterior ends terminating at posterior margins of ear openings, edged dorsally and ventrally by wide dark-brown margin, yellow. Only two complete body bands can be recognized between nuchal loop and caudal constriction: first band located posterior to axilla; second band inserts onto dorsal surface of thigh, bands on limbs dirty yellow, lacking dark spots, edged by broad dark-brown borders anteriorly and posteriorly, other blotches incomplete, not forming a complete bands. Supralabials and infralabials grayish brown; pupils vertical and appear black; iris orange, gradually darkening on both sides; dorsal surface of limbs light grayish brown with dark brown and dirty yellow tubercles and dark spots and blotches; chin, throat, thorax, and ventral surfaces of body pink, tinged brownish, with dark-brown lateral spots; ventral surface of limbs pink, tinged brownish, without dark-brown spots; digits light grayish brown; ground color of original tail dark brown with nine immaculate white caudal bands completely encircling the tail, and a white tip. Body color becomes darker after capture.
Coloration in preservative. Dorsal ground color of head, body, and limbs become darker; ventral surface faded to grayish white; all darker spots, blotches and bands on dorsal surface blurred.
Variations. Measurements of type series specimens are shown in Table 3. Two paratypes have same approximate measurements as holotype, but have significant variations in coloration. Male paratype (SYS r002421, Fig. 3B) has four body bands, the second band extending a three forked branch connecting with third band. Female paratype (SYS r002422, Fig. 3C) has three complete body bands, second band extending a branch forward to middle of body, original tail has ten immaculate white caudal bands completely encircling tail, and dark-brown tip.  Data come from Grismer et al. 1999Grismer et al. , 2002Orlov et al. 2008;Ziegler et al. 2008;Blair et al. 2009;Wang et al. 2010Wang et al. , 2013Wang et al. , 2014Yang and Chan 2015;Zhou et al. 2018;Qi et al. 2020;Zhu et al. 2020. Etymology. The specific epithet "gollum" is named after the fictional character, Gollum, from J.R.R. Tolkien's The Lord of the Rings book series. This new species and Gollum have similar cave-dwelling habit and emaciated body. We suggest the common name as "Gollum Leopard Gecko", and according to the type locality, we suggest the Chinese formal name as "guǎng dōng jiǎn hǔ" (广东睑虎).

Character
Distribution and ecology. Currently, Goniurosaurus gollum sp. nov. is known only from Huaiji County, Guangdong Province, China. All individuals were found within a barren limestone cave approximately 50 m from the cave entrance at night after 2130 hrs (Fig. 5). The surface of the cave is covered with bat (unidentified) and bird (Apus pacificus) droppings. Stalactites are suspended from the roof and there is no vegetation. Duttaphrynus melanostictus was observed in the same area.

Discussion
With the description of Goniurosaurus gollum, there are now 23 known species in the eublepharid genus Goniurosaurus, 15 of which occur in China. The G. yingdeensis group is endemic in Guangdong Province, with only two species recognized previously, but the discoveries of G. varius (Qi et al. 2020) and G. gollum adds to this and indicates there might be more new species yet to be discovered.
Although the species of Goniurosaurus are also called "cave geckos", they prefer inhabiting the forest floor, limestone cliffs, rocky and cement drains along the road, and near the entrances of caves. However, the cave-dwelling species G. gollum appears to be a true cave dweller as opposed to other Goniurosaurus. The fact that all three specimens were found at least 50 m inside the cave from its entrance supports this hypothesis.
These results support the growing body of evidence from China (Luo et al. 2016) and elsewhere (Grismer et al. 2016 and references therein) that karstic habitats not only provide a substrate for the evolution of new species-especially gekkotan lizards-but maintain levels of biodiversity that rival that of other tropical habitats. Yet they are some of the most imperiled and least protected habitats in the world (Day and Urich 2000;Gillieson 2005). The discovery of yet another new species in the vast karstic landscape of northern Guangdong underscores this fact and brings into sharp focus the urgent need to protect these unique landscapes. We aim to protect G. gollum from the pet trade, by withholding its precise locality. However, these data are available to qualified researchers upon official request to The Museum of Biology, Sun Yat-sen University.