A new species of Cyrtodactylus Gray, 1827 (Squamata, Gekkonidae) from Yunnan, China

Abstract A new species of Cyrtodactylus is described on the basis of five specimens collected from the karst formations of Zhenkang County, Yunnan Province, China. Cyrtodactylus zhenkangensissp. nov. is recognized by having a unique combination of morphological characters, the most diagnostic being: 12–15 enlarged femoral scales on each thigh; 2–5 femoral pores on each thigh in males, 0–3 pitted scales on each thigh in females; eight or nine precloacal pores in a continuous row or separated by one poreless scale in males, 7–9 pitted scales in females; subcaudals enlarged, arranged alternately as single and double on anterior and mostly single at middle and posterior; dorsal surface of head with obvious reticulations. Phylogenetic analyses show that the new species is a member of the C. wayakonei species group and a sister taxon to a clade consisting of C. wayakonei and C. martini based on Maximum Likelihood analyses and Bayesian Inference and differs from its congeners by at least 12.0% genetic divergence in a fragment of the COI gene.


Introduction
Bent-toed geckos of the genus Cyrtodactylus are one of the most species-diverse genera of gekkonid lizards (Kluge 2001;Uetz 2020), and many of these species are thought to be highly localized with extremely narrow geographic ranges (Nazarov et al. 2012;Luu et al. 2016;Grismer et al. 2018Grismer et al. , 2020Murdoch et al. 2019). At present, the genus contains more than 300 recognized species (Uetz et al. 2020), and approximately 150 new species have been described since 2010 and most of these new discoveries were from Southeast Asia (Schneider et al. 2020).
During our recent fieldwork in Yunnan Province, China, a series of bent-toed geckos was collected from the karst formations of Zhenkang County. Morphological and molecular phylogenetic analyses revealed that the new collection belonged to an unnamed species of Cyrtodactylus. We describe it as a new species.

Sampling
Fieldwork was conducted at night. Specimens were collected by hand. Photographs were taken to document color pattern in life prior to euthanization. Liver tissues were stored in 99% ethanol and specimens were preserved in 75% ethanol. Specimens were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Molecular analyses
Molecular data were generated for three specimens and analyzed with the available homologous sequences of the Cyrtodactylus wayakonei species group obtained from GenBank. The new sequences were deposited in GenBank under accession numbers MW593136-MW593138. Sequences of C. cf. interdigitalis Ulber, 1993 and C. elok Dring, 1979 were used as outgroups according to Nguyen et al. (2017) and Schneider et al. (2020).
We used the protocols of Le et al. (2006) for DNA extraction, amplification, and sequencing. DNA extraction used the standard three-step phenol/trichloromethane protocol (Sambrook et al. 1989). A fragment of the mitochondrial gene, cytochrome c oxidase subunit 1 (COI) was amplified in a volume consisted of 25 μl (10 μl of mastermix, 5 μl of water, 2 μl of each primer at 10 pmol/μl and 6 μl of DNA) by the polymerase chain reaction (PCR; 35 cycles of 95 °C for 30 s, 53 °C for 40 s, 72 °C for 90 s) and sequenced using the primer pair VF1-d (TTCT-CAACCAACCACAARGAYATYGG) and VR1-d (TAGACTTCTGGGTGGC-CRAARAAYCA) (Ivanova et al. 2006). PCR products were cleaned using ExoSAP-IT (Applied Biosystems) and sequenced in both directions by direct double strand cycle sequencing using the BigDye Terminator v. 3.1 Cycle Sequencing Kit on a 3130 DNA Analyzer (Applied Biosystems). Sequences were edited with Sequencher v. 5.4.6 (Gene Codes).
Sequences were aligned using ClustalW (Thompson et al. 1994) integrated in MEGA v. 7 (Kumar et al. 2016) with default parameters. Pairwise distances between species were calculated in MEGA v. 7 with the parameters Transitions + Transversions, Uniform rates, and Pairwise deletion (Kumar et al. 2016). The substitution model GTR+G+I was selected using the corrected Akaike Information Criterion (AICc) in MODELTEST v. 3.7 (Posada and Crandall 1998). Bayesian inference (BI) was performed in MrBayes v. 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 10,000,000 generations and sampled every 1000 generations. The first 25% of the sampled trees was discarded as burn-in after the standard deviation of split frequencies of the two runs reached a value of less than 0.01, and then the remaining trees were used to create a 50% majority-rule consensus tree and to estimate Bayesian posterior probabilities (BPP). Nodes with BPP of 95 and above were considered strongly supported (Huelsenbeck et al. 2001;Wilcox et al. 2002;Alfaro et al. 2003) and nodes with values of 90-94 as well supported (Chomdej et al. 2020). Maximum Likelihood (ML) analysis was performed in RaxmlGUI v. 1.5 (Silvestro and Michalak 2012), and nodal support was estimated by 1,000 rapid bootstrap replicates. Nodes with bootstrap values of 70 and above were considered significantly supported (Alfaro et al. 2003;Sitnikova 1996).

Molecular analyses
The obtained sequence alignment is 690 bp in length. The topologies derived from ML and BI analyses were similar and basically consistent with those of Nguyen et al. (2017), Pham et al. (2019), andSchneider et al. (2020). The sequences of three specimens collected from Zhenkang County, Yunnan, China were nested them within the Cyrtodactylus wayakonei group and the sister group to a clade consisting of C. wayakonei Nguyen, Kingsada, Rösler, Auer &Ziegler, 2010 andC. martini Ngo, 2011 with strong support in ML and moderate support in BI (Fig. 1). The interspecific uncorrected genetic p-distances between the newly collected specimens and other members of C. wayakonei group ranged from 12.0% to 17.8% (Table 1).

Figure 1.
Bayesian Inference phylogram inferred from partial COI genes. Numbers before slashes indicate bootstrap support for Maximum Likelihood analyses and numbers after slashes indicate Bayesian posterior probabilities. The symbol "-" represents the value below 60.
Paratypes. KIZL2020048 and KIZL2020050, two adult females; KIZL2020046, subadult male; and KIZL2020047, subadult female; all the same collection data as the holotype.
Etymology. The name refers to Zhenkang County, where the new species was found. Diagnosis. Cyrtodactylus zhenkangensis sp. nov. differs from all other congeners by the following combination of characters: medium size (SVL 78.1-87.4 mm); ventrolateral folds present with interspersed tubercles; 12-15 enlarged femoral scales on each thigh; 2-5 femoral pores on each thigh in males, 0-3 pitted scales on each thigh in females; eight or nine precloacal pores in a continuous row or separated by one poreless scale in males, 7-9 pitted scales in females; two or three postcloacal tubercles on each side; 18-21 lamellae under finger IV, 21-23 lamellae under toe IV; subcaudals enlarged, arranged alternately as single and double on anterior and mostly single at middle and posterior; dorsal surface of head with obvious, light-colored reticulations; eight or nine irregular transverse bands on the dorsum of body.

Color of holotype in life.
Head brown with pale-yellow, slightly symmetrical reticulations on either side of the midline, no dark-colored nuchal loop; dorsum of body brown with approximately nine pale-yellow, transverse, irregular bands from forelimb insertions to base of tail and one longitudinal, continuous, narrow vertebral stripe; dorsal surface of limbs brown with some light-yellow, irregularly shaped bands, some small, light-yellow spots on the dorsum of fingers and toes; ventral surface of head, body, and limbs grey with no stripes or spots; tail brownish black with ten yellowish white rings; iris copper-yellow.
Variations. Color pattern variations are shown in Figure 5, and morphometric and meristic differences are presented in Table 2. Morphologically the paratypes resemble the holotype except as follows: KIZL2020046 and KIZL2020047 each has one vertebral stripe like the holotype but it is discontinuous; KIZL2020050 has one continuous vertebral strip and two discontinuous, longitudinal, narrow stripes on the sides of vertebral strip; KIZL2020048 only has transverse bands and no vertebra stripe. All paratypes have continuous precloacal pores (pitted) and fewer femoral pores (pitted).
Distribution. The new species is currently known only from the type locality in Zhenkang County, Yunnan Province, China.
Natural history. All specimens were found at night between 19:00 and 21:00 on limestone cliffs of the karst formations. The surrounding habitat was primary forestwith a stream nearby. No eggs or juveniles were found.
Cyrtodactylus zhenkangensis sp. nov. differs from C. martini in having femoral pores in males (vs lacking femoral pores in males) and more irregular transverse bands on the dorsum of body (8-9 vs 5-7).
For other species which were not included in the phylogenetic analyses and resemble Cyrtodactylus zhenkangensis sp. nov. in morphology. Cyrtodactylus zhenkangensis sp. nov. differs from C. auribalteatus Sumontha, Panitvong & Deein, 2010 in having more transverse bands on the dorsum of body (8-9 vs 4-5), obvious reticulations on the dorsum of head (vs no obvious reticulations) and absent dark-colored nuchal loop (vs present).
Although the distribution of the new species is distant from the distributions of C. martini and C. wayakonei, the new species is most similar to the latter two in both morphology and phylogeny. The new species is not found in a protected area; the type locality is just beside the county seat, where there are human activities during the day but usually not at night. This species is nocturnal, so it may be less affected by human activities.
There are many other karst formations in Yunnan, some of which remain insufficiently surveyed. We are continuing to conduct more expeditions in these regions, and it is likely that additional new species of Cyrtodactylus will be found in these karst systems.