First record of Lepidodactylus lugubris (Duméril & Bibron, 1836) (Squamata, Gekkonidae) from Hainan Island, China

Lepidodactylus lugubris is known from tropical Asia, Oceania, and Latin America, but in China it was previously known only from Taiwan Island. In this paper, we report a new herpetofaunal record based on one specimen collected from Wanning, Hainan, China, which conforms to L. lugubris on both morphological and molecular data. Our finding brings the total species of the family Gekkonidae in Hainan to six.


Introduction
The genus Lepidodactylus Fitzinger, 1843, currently contains 45 species that are distributed from Southeast Asia to Indo-Australia and Oceania, the majority of which are endemic to, or with narrow-range in, tropical islands (Uetz et al. 2022). By contrast, the most wide-ranging species of this genus is undoubtedly the mourning gecko Lepidodactylus lugubris (Duméril & Bibron, 1836). It has spread to tropical Asia, Oceania, and Latin America (introduced) from tropical Indo-Pacific islands (where it is thought to be native) in past decades, due to parthenogenetic reproduction and resistance of eggs to desiccation and saltwater spray (Cuellar and Kluge 1972;Brown and Duffy 1992;Bauer and Henle 1994;Hoogmoed and Avila-Pires 2015;Lapwong and Juthong 2018;Urra et al. 2020). The parthenogenetic lizard is comprised of several diploid and triploid clones that originated by hybridization between Lepidodactylus moestus (Peters, 1867) and a species that was undescribed until recent research concluded that it was possibly Lepidodactylus pantai Stubbs, Karin, Arifin, Iskandar, Arida, Reilly, Bloch, Kusnadi & McGuire, 2017(Radtkey et al. 1995Fujita and Moritz 2010;Ineich 2015;Karin et al. 2021). Lepidodactylus lugubris is a nocturnal species usually found near the coast, inside houses or on trees (Señaris et al. 2017;Behm 2018;Nania et al. 2020). Its spread is often thought to be possibly related to human activity, such as artificial transportations and cargo shipping (Kraus 2009;Nania et al. 2020). In China, L. lugubris was previously known only from Taiwan Island as a controversial native species (Ota 1986;Lee et al. 2019).
During our field survey from April to December 2021, a gekkonid lizard with a small and elongated body was collected in Hainan Island, China. Morphological comparison and molecular analysis indicated that this individual belongs to the species Lepidodactylus lugubris. Herein, we report this new herpetofaunal record from Hainan Island, China in detail.

Materials and methods
Field surveys were conducted in Shimei Gulf, Wanning City, Hainan Province. The specimen was euthanized and then fixed in 10% buffered formalin, later transferred to 75% ethanol. It is deposited in Shenyang Normal University (SYNU), Shenyang, China. Liver tissue sample was preserved in 95% ethanol for molecular analysis.
Genomic DNA was extracted from liver tissue using a DNA extraction kit (Tiangen Biotech Co., Ltd, Beijing). The mitochondrial fragment of the NADH dehydrogenase subunit 2 gene (ND2) sample was sequenced using primer rMet-3L (5'-ATACCCCGACAATGTTGG-3') and rAla-1H (5'-GCCTTAGCTTAATTAAAGTG-3') (Jonniaux and Kumazawa 2008). The PCR procedure was performed with an initial denaturation at 95 °C for 5 min, 35 cycles of 95 °C for 40 s, 53 °C for 40 s and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. PCR products were purified with spin columns and then sequenced with a forward primer using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA). Sequencing was performed on an ABI Prism 3730 automated DNA sequencer by Wuhan Tianyi Huiyuan Bioscience and Technology Inc.
Measurements were taken following Zhao et al. (1999) and Kraus and Oliver (2020). All external measurements were made using digital calipers to the nearest 0.1 mm: SVL (from tip of snout to vent); TrL (trunk length from posterioredge of forearm insertion to anterior edge of hindleg insertion); TL (from vent to tip of tail); TW (maximum tail width); HL (head length from tip of snout to anterior margin of ear opening); HW (maximum head width); HH (maximum head height); FA (forearm length from central base of palm to elbow); CS (crus length from central base of heel to knee); Ear (maximum ear diameter); EE (shortest ear-to-eye distance); EY (greatest eye diameter); EN (eye-naris distance from anteriormost point of eye to posteriormost point of naris); SN (snout length from anteriormost point of eye to tip of snout); IN (internarial distance between centers of nares); EE (shortest ear-to-eye distance); RW (maximum rostral width); RH (maximum rostral height); MW (maximum mental width); ML (maximum mental length); T4L (length of the fourth toe from terminal lamellae to the beginning of the web between T3 and T4); T4W (maximum width of the fourth toe); T4la-mellaeL (length of the series of lamellae on the fourth toe); T3T4webL (length of webbing between T3 and T4 from base of this webbing to its centre of emargination) and T4T5webL (length of webbing between T4 and T5 from base of this webbing to its center of emargination). In addition, scalation features include supralabials, infralabials, lamellae under T1 and T4, and divided lamellae under T4. Bilateral scale counts are given as left/ right.

Phylogenetic analysis
Twenty-one sequences from seven known Lepidodactylus species plus one out-group sequence from the gekkonid Hemiphyllodactylus huishuiensis which was used to root the tree were obtained from GenBank and comprised the dataset (Table 1). Sequences were aligned in Clustal X 2.0 (Thompson et al. 1997) with default. The alignment was checked and manually revised if necessary. Gaps/ Missing Data Treatment use the complete-deletion option in MEGA 6.06, substitutions to included: Transitions + Transversions option (Tamura et al. 2013). We ran Jmodeltest v2.1.2 (Darriba et al. 2012) with Akaike and Bayesian information criteria on the alignment, resulting the best-fitting nucleotide substitution models of GTR + I + G. Phylogenetic analysis was constructed using Bayesian inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012). Two independent runs with four Markov Chain Monte Carlo simulations were performed for ten million iterations and sampled every 1000 iterations. The first 25% of samples were discarded as burn-in. Convergence of the Markov Chain Monte Carlo simulations was assessed with PSRF ≤0.01 and ESS (effective sample size) value > 200 using Tracer 1.4 (http://tree.bio.ed.ac. uk/software/tracer/). Uncorrected pairwise sequence divergences utilizing the ND2 gene were calculated using MEGA 6.

Results
The ML and BI analyses resulted in identical topologies (Fig. 1). Uncorrected pairwise sequence divergences are reported in Table 2. As illustrated by the phylogenetic result, the Lepidodactylus sample from Hainan, China is clustered together with other Lepidodactylus lugubris samples and exhibit robust monophyletic lineage with strong support (BPP 1.00) and low intrapopulational genetic differentiation (0.13-0.76%; Table 2). Thus, we regard this specimen as representing a new herpetofaunal record from Hainan Island, China.
Morphological description. Morphological characters of the specimen agreed well with the Duméril and Bibron's original description (translated by Amarasinghe et al. 2009) and subsequent description of Zhao et al. (1999) in Chinese.
Coloration. In life, dorsal surface of head reddish brown; a narrow dark brown stripe along the canthus rostralis, crossed the eye, to near shoulder; the major colors of the body, limbs and tail are yellowish sepia, as is the head; a series of indistinct W-shaped markings down the center of the neck and tail, each angular base of the W usually with a small blackish spot, especially the markings at the neck and the base of tail; dark brown stripes all over the dorsal limbs; some light and dark spots on dorsal tail; ventral surface of head milk white, ventral body coverd by lemon yellow band, with brownish spots on each side; ventral tail light brown in forepart and gradual deepening towards the end with dark brown spots ( Fig. 2A, B).
In preservative, dorsal surface of specimen grayish white, ventral surface discolored to milky white; the W-shaped patterns indistinct, leaving mostly black bars (Fig. 3A, B).
Distribution and ecological notes. Currently L. lugubris is known from almost all over tropical Asia (China, Sri Lanka, India, Myanmar, Malaysia, Vietnam, Japan, Indonesia etc.), Indo-Pacific (Christmas Island)and Oceania (Fiji Islands, Rotuma, New Caledonia, Loyalty Islands, Vanuatu, Tonga etc.), even introduced into the continent of the Americas and the Caribbean (Behm 2018;Urra et al. 2020;Uetz et al. 2022). In China, this species is only known on Hainan Island (Wanning City) and eastern and southern Taiwan Island (including Orchid Island and Green Island; Lee et al. 2019).
The specimen was found at 20:30 on a banyan tree about 1.5m above the ground near a coastal beach (Fig. 2C). Two opal eggs were visible through the skin of the belly.

Discussion
The discovery of the new record brings the total species of Gekkonidae in Hainan Province to six. The five species known from the province are Gekko ( The species L. lugubris actually contains several diploid and triploid clones, in which diploid clone A and triploid clone C are similar and confused on morphology (Ineich 1988(Ineich , 1999. Based on previous studies, Ineich (2015) summarized the morphological differences between clone C and other clones when studying samples from the Wallis and Futuna Islands. The main morphological differences include the black stripes on the sides of the W-shape on the neck that never disappeared and the ventral surface covered by lemon yellow band. By these two characteristics, we can easily determine that the Hainan specimen belongs to clone C (Fig. 2). This clone is widespread but uncommon (Ineich 2015). Hence, the origin of L. lugubris in Hainan Island is awaiting further study. Meanwhile, given that the species has not been found on the island for a long time and that it is a well-known invasive species, we tend to consider it a non-native species. Nania et al. (2020) reconstructed the geographic expanding history of L. lugubris and predicted further expansion of its geographic range over time, in which Hainan Island was considered one of the suitable areas for the species. Although the existence of stable populations in Hainan Island needs to be surveyed once again, our findings tentatively support this hypothesis. to Robert W. G. Jenkins for his helpful comments on this manuscript. We also thank Dr. Peter Mikulíček and two anonymous reviewers for their constructive comments and suggestions on the manuscript. This work was supported by Hainan Shangxi Amphibian and Reptile Monitoring Program.