A definition of the Goniurosaurus yingdeensis group (Squamata, Eublepharidae) with the description of a new species

Abstract A definition of the Goniurosaurus yingdeensis group is presented in this study, on the basis of morphological and phylogenetic analyses based on a series of additional specimens. Moreover, a new species of this group, Goniurosaurus variussp. nov., is proposed for northern Guangdong Province, China. The new species can be distinguished from the other two congeners of this group by the following unique characters: one or two internasals; enlarged supraorbital tubercles absent; paravertebral tubercles between limb insertions 27–29; dorsal tubercle rows at midbody 21–24; ten precloacal pores in males and absent in females; body bands with black spots; iris orange-red.

During the herpetological surveys conducted from 2015 to 2019, a number of Goniurosaurus specimens were collected from northern Guangdong Province, China that should be placed in the G. yingdeensis group on the basis of both morphological and molecular analyses. Furthermore, these specimens can be distinguished from congeners by discrete morphological differences and genetic divergences, and represent an unidentified taxon within the G. yingdeensis group. In the present study, this taxon is described as a new species and the Goniurosaurus yingdeensis group is revised and defined.

Sampling
Sixteen specimens of Goniurosaurus yingdeensis were collected from the Shimentai National Nature Reserve, Yingde City, Guangdong Province (including six type specimens) for morphological comparison, and four specimens (SYS r001271, 1272(SYS r001271, , 1493(SYS r001271, , 2115 were used in the phylogenetic analysis. Nine specimens of G. zhelongi were collected from the Shimentai National Nature Reserve, Yingde City, Guangdong Province (including five type specimens) for morphological comparison, and four specimens (SYS r000816, 1491(SYS r000816, , 1492(SYS r000816, , 2108 were used in the phylogenetic analysis. Five specimens of the undescribed species were collected from the Nanling National Nature Reserve, Chengjia Yao Ethnic Township, Yangshan County, Guangdong Province, China, and all of them were used in phylogenetic analysis. Following euthanasia, all specimens were fixed in 10% formalin and transferred to 75% alcohol; they are deposited in the Museum of Biology, Sun Yat-sen University (SYS), Guangdong Province, China.
Due to the cryptic diversity in genus Goniurosaurus, we choose sequences from type series or topotype specimen for molecular analysis if available, to ensure the taxonomic identity of the species being studied. A total of 10 samples from four known species (one sample of Goniurosaurus bawanglingensis, four samples of G. yingdeensis, three samples of G. zhelongi, and two samples of G. zhoui) and five samples of the unidentified species were used. Tissues samples were taken before the specimens were fixed in 10% formalin, preserved in 99% alcohol, and stored at -40 °C. Sequences of other species of Goniurosaurus follow Liang et al. (2018); for details see Table 1.

Species delimitation
The general lineage concept (GLC; de Queiroz 2007) adopted herein proposes that a species constitutes a population of organisms evolving independently from other such populations owing to a lack of gene flow. By "independently", it is meant that new mutations arising in one species cannot spread readily into another species (Barraclough et al. 2003;de Queiroz 2007). Integrative studies on the nature and origins of species are using an increasingly wide range of empirical data to delimit species boundaries (Coyne and Orr 1998;Fontaneto et al. 2007;Knowles and Carstens 2007;Leaché et al. 2009), rather than relying solely on morphology and traditional taxonomic methods. Under the GLC implemented herein, molecular phylogenies were used to recover monophyletic mitochondrial lineages of individuals (populations) in order to develop initial species-level hypotheses -the grouping stage of Hillis (2019). Discrete color pattern data and univariate and multivariate analyses of morphological data were then used to search for characters and morphospatial patterns bearing statistically significant differences that were consistent with the previous designations of the species-level hypotheses, the construction of boundaries representing the hypothesis-testing step of Hillis (2019), thus providing independent diagnoses to complement the molecular analyses.

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 eyeto-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 orbit); GST granular scales surrounding dorsal tubercles; PTL paravertebral tubercles between limb insertions; DTR dorsal tubercle rows at midbody; MB scales around midbody; PP precloacal pores; PAT postcloacal tubercles. Bilateral scale counts are given as left/right.
Data of characters 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) and 30 museum specimens of the seven species listed in the Appendix 1 were examined.

Phylogenetic analyses
Twenty sequences from eleven known Goniurosaurus species and one out-group sequence from Hemitheconyx taylori in the Eublepharidae used to root the tree, were obtained from GenBank and incorporated into our 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 482 base pairs (bp) of 16S and 396 bp of Cytb, were concatenated seriatim into an 878 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 Mr-Bayes 3.2.4 (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. Uncorrected pairwise sequence divergences utilizing the 16s gene were calculated using MEGA 6 (provide ref for MEGA 6).

Statistical analyses of morphology
An analysis of variance (ANOVA) was conducted on characters with statistically similar variances (i.e., p values ≤ 0.05 in a Levene's test) to search for the presence of statistically significant mean differences (p < 0.05) across the data set. Characters bearing statistical differences were subjected to a TukeyHSD test to ascertain which population pairs differed significantly from each other for those particular characters. The mensural characters were scaled to SVL in order to remove any potential effects of allometry using the following equation: X adj = log(X)-β[log(SVL) -log(SVL mean )], where X adj = adjusted value; X = measured value; β = unstandardized regression coefficient for each population; and SVL mean = overall average SVL of all populations (Thorpe 1975(Thorpe , 1983Turan 1999;Lleonart et al. 2000). Boxplots were generated in order to visualize the range, mean, and degree of differences between pairs of species bearing statistically different mean values for sets of characters.

Results
The ML and BI analyses resulted in essentially identical topologies (Fig. 1). Uncorrected pairwise sequence divergences are reported in Table 2. The phylogenetic analyses showed that Goniurosaurus can be divided into four strongly supported clades consistent with the recognition of the four species groups proposed by Liang et al. (2018), i.e., the G. kuroiwae group, G. lichtenfelderi group, G. luii group, and G. yingdeensis group.
The Goniurosaurus yingdeensis group is divided into three subclades with moderate genetic differences among them (3.3-4.7%), two of which represent G. yingdeensis and G. zhelongi, respectively; the third subclade is composed of the new population from northern Guangdong Province with a high nodal support value (1.00 in BI and 100% in ML) and low intrapopulational genetic differentiation (0-0.3%) and represents an unnamed species of Goniurosaurus (Table 2). Additionally, this population has a combination of characteristics (see below) distinguishing it from other species in the G. yingdeensis group while showing significant differences from all known congeners. ANOVAs and subsequent TukeyHSD tests recovered significantly different mean values among various combinations of species across various combinations of characters (Tables 3, 4; Figs 2, 3).
Based on phylogeny and corroborating statistically significant differences in morphology (Figs 1-3), we propose that the northern Guangdong Province population is a new species of the Goniurosaurus yingdeensis group. The discovery of this new species has provided valuable new morphological and genetic information on this species group. The previous designation of the Goniurosaurus yingdeensis species group was on the based solely on molecular data and lacked a morphological definition. Therefore, along with the description of a new species, we provide the first morphological definition of the Goniurosaurus yingdeensis group.

Class Reptilia Laurenti, 1768
Order Squamata Oppel, 1811 Family Eublepharidae Boulenger, 1883 Genus Goniurosaurus Barbour, 1908 Goniurosaurus yingdeensis group Morphological definition. This species group can be differentiated from the other species groups by the combination of the following characters: (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 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.
Comparison. The Goniurosaurus yingdeensis group can be distinguished from the three other known species groups by the base of claws being sheathed by four scales, two lateral scales of claw 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 by four scales in G. kuroiwae group; precloacal pores less than 15 in males vs. 17-46 in G. lichtenfelderi group (37-46 in G. bawanglingensis, 24-32 in G. hainanensis, 17-32 in G. lichtenfelderi, 36-38 in G. zhoui), 16-33 in G. luii group (18-22 in G. araneus, 16-21 in G. catbaensis, 25-28 in G. huuliensis, 26-28 in G. kadoorieorum, 31-33 in G. kwangsiensis, 23-28 in G. liboensis, 23-29 in G. luii) and absent in G. kuroiwae group.   Summary statistics of the species of the Goniurosaurus yingdeensis group are listed in Table 4. Additional comparisons of morphological characteristics are provided in Table 5   (SYS r000536) and a juvenile female (SYS r000552). All specimens collected from the Shimentai National Nature Reserve.
Variation. Overall morphology, coloration, and scalation data of the newly discovered populations of G. yingdeensis are in general agreement with the description of the holotype by Wang et al. (2010). Males have 10-13 distinct precloacal pores, whereas precloacal pores are present but indistinct in two adult females (SYS r000535, SYS r001652) and a subadult female (SYS r000536), absent in another two adult females (SYS r001271, SYS r001272); internasal usually numbering two or three, but single in the two females (SYS r001271, SYS r001652). Additional variation in scale counts and measurements are shown in Table 6. For female precloacal pores see Fig. 6. Diagnosis.
Variation. Overall morphology, coloration, and scalation data of the newly discovered populations of G. zhelongi are in general agreement with the description of the holotype by Wang et al. (2014). Precloacal pores usually nine in males, 12 in an adult male (SYS r001491) and absent in female; internasal single or two. Additional variation in scale counts and measurements are shown in Table 7. Diagnosis.

Goniurosaurus varius
Etymology. The specific name varius means varied or diverse in Latin and refers to its variable dorsal color pattern. As the type locality locates within the Nanling National Nature Reserve, we suggest the common name as "Nanling Leopard Gecko".
Description of holotype. An adult male with regenerated tail; SVL 84.7 mm; HL 22.7 mm; HW 16.0 mm; SE 9.1 mm; EE 13.0 mm; SVL:HL 3.7; HL:HW 1.4; SE:EE 0.7. 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 with almost uniform size; scales of rostrum slightly larger than those in between orbits; rostral convex and hemi-elliptic, 1.3 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 7/8 nasals each, anteriorly by prenasal and supranasal, dorsally by supranasal and a granular scale, posteriorly by 5/5 smaller granular scales, and ventrally by the prenasal; prenasal with long recurved ventral portion; supranasals large, separated by one internasals; supralabials rectangular, 8/10; preorbital scales 15/15; eyes relatively large, pupils vertical; eyelid fringe scales 50/52; outer surface of upper eyelid composed of granular scales of about the 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, 9/9; 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 105; dorsal tubercle rows at midbody 24; vertebral row of scales indistinct; paravertebral tubercles between limb insertions 27; dorsal body tubercles surrounded by 9-11 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 the level of the vent.
Regenerated tail, short, thin at base, gradually thickening posteriorly, and gradually thinning into an obtuse tip; dorsal scales in regenerated portion of tail flattened, subimbricate, arranged in more or less regular transverse rows; subcaudal scales flattened, smooth, subimbricate, slightly larger than dorsal caudal scales.
Limbs relatively long and slender; dorsal surface covered with granular scales, densely interspersed with tubercles; ventral surface covered by flat scales, juxtaposed, 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 shell-shaped. Coloration in life. Dorsal ground color of head, neck, body, and limbs reddish brown, mottled with indistinct faint dark brown-colored markings, scattered with densely light yellow tubercles and a few dark brown and reddish brown tubercles; nuchal loop incomplete, just from posterior corner of eyes to the temporal region, dirty yellow; four narrow body bands between the nuchal loop and the caudal constriction, fourth band inserting into the dorsal thigh, bands dirty yellow, with dark spots, edged in dark-brown anteriorly and posteriorly; a longitudinal light colored vertebral stripe between third and fourth bands; supralabials and infralabials grayish brown; pupils vertical and black; iris orange-red; dorsal surface of limbs deep reddish brown with dirty yellow tubercles and indistinct dark spots; chin, throat, thorax, and ventral surfaces of body pink, tinged brownish, with dark-brown lateral spots; ventral surface of limbs pink, tinged brownish, with dark-brown spots; digits gray; ground color of the regenerated tail dark-brown, one original white band on the bases of tail, followed by irregularly shaped white markings. The body color becomes darker after capture.
Coloration in preservative. Dorsal ground color of head, body, and limbs black; ventral surface faded to grayish white; all darker spots and bands on the dorsal surface blurred.
Coloration in juvenile. Dorsal ground color of head, neck, body, and limbs darkorange, mottled with indistinct faint dark-brown-colored markings, scattered with dense light yellow tubercles and a few dark-brown tubercles; nuchal loop from posterior corner of the mouth to the back of head, light yellow; four narrow body bands between the nuchal loop and the caudal constriction, fourth band inserting into the dorsal thigh, band color light yellow with dark spots, edged in dark-brown anteriorly and posteriorly (but not laterally); supralabials and infralabials grayish brown; pupils vertical and black; iris orange-red; dorsal surface of limbs dark orange with orange tubercles and indistinct dark spots; chin, throat, thorax, and ventral surfaces of body pink; ventral surface of limbs pink with dark-brown spots; digits gray; tail black-grey bearing white caudal bands encircling tail.
Variations. Measurements of type series specimens are shown in Table 8. Three paratypes have more complete and distinct nuchal loops than holotype, but SYS r002363 has only half a nuchal loop from the posterior corner of the right eye to the back of head. SYS r002330 has vertebral stripe extending from the posterior edge of the second body band to the anterior edge of third body band. SYS r002331 and SYS r002363 have large dark dorsal blotches on the head and the body band margin are broader than those in the holotype. Also, SYS r002363 has immaculate body bands. An additional female specimen (Fig. 10B) shows a more mottled dorsal pattern than all other types and its bands are mingled with irregular patterns on the body. SYS r002362 (Fig. 10C) has smaller dorsal blotches making it appear almost as if it has a reticulated dorsal pattern and its bands are greatly obscured, it has a distinct white vertebral stripe from the posterior edge of the first body band extending to the anterior edge of the last body band.
Distribution and ecology. Goniurosaurus varius sp. nov. is currently known only from the karst environment of the Nanling National Nature Reserve, northern Guangdong Province, China ( Figure 11). All individuals were found in crevices of limestone near villages, farmlands, or country lanes at elevations between 180 and 560 m at night after 21:00 hrs.

Discussion
Our continued herpetological surveys coupled with extensive sampling in Guangdong Province, China in the past decades have resulted in discovery of three new species of Goniurosaurus from two localities, which all belong to the G. yingdeensis group. Topographically, rivers and a canyon form a series of geographic barriers that might lead to the isolations of members of G. yingdeensis group. Among them, G. yingdeensis is distributed in the lower hill areas on the east side of the Ruyuan Canyon, G. zhelongi was found on the west side of canyon. Moreover, microhabitat selection might also play an important role in species differentiation. Nearly all of G. varius individuals were found in karst topography but G. yingdeensis and G. zhelongi were also found in granitic landforms. This suggests they may be saxicolous generalist as opposed to a microhabitat specialist. Future phylogeographic and habitat selection studies are needed to gain a better understanding of their evolutionary history.
As the development of integrated taxonomy, to combine the morphological comparisons and phylogenetic relationships, has become an important and necessary work. In the present study, we propose the morphological definition of the Goniurosaurus yingdeensis group, which can be significantly distinguished from all other congeners, consistent with their distinct divergences in phylogeny. Nevertheless, it is worth noting that the species G. bawanglingensis and G. zhoui can be assigned to the G. luii group according to previous morphological diagnoses (Grismer et al. 2002;Zhou et al. 2018), while they were clustered within G. lichtenfelderi group in phylogeny based on two mitochondrial and two nuclear genetic segments . Hence, further comprehensive work with detailed morphological examinations and more genetic data is asked for, to clarify these incongruences or revise the morphological definitions of the G. luii group and the G. lichtenfelderi group.