Hiding in the valley: a new national record of Ablepharus eremchenkoi , with rediscovery of Ablepharus alaicus in China: phylogeny, morphology and natural history notes

* These authors


Introduction
The Ablepharine is a widespread skink lineage distributed from Europe to Asia (Vaissi et al. 2023).Asymblepharus Eremchenko &Shcherbak, 1986 andHimalblepharus Eremchenko, 1987 were established as separate genera from Ablepharus Lichtenstein, 1823, based on morphological divergence.In a recent revision of Ablepharine skinks, Mirza et al. (2022) proposed treating Asymblepharus and Himalblepharus as subjective junior synonyms of Ablepharus because both are embedded within Ablepharus, which does not have a movable eyelid.This returns species belonging to these two genera to Ablepharus sensu lato, including Asymblepharus alaicus, which are mainly distributed in Central Asia in Kyrgyzstan, Uzbekistan, Tajikistan, Kazakhstan and China.
There are three subspecies of A. alaicus: A. a. alaicus Elpatjevsky, 1901;A. a. kucenkoi Nikolsky, 1902;andA. a. yakovlevae Eremchenko, 1983. Panfilov (1999) evaluated some populations of A. a. yakovlevae as A. eremchenkoi, based on variations in reproductive traits.Two subspecies were thought to be distributed in China (Zhao et al. 1999).Of these, A. a. kucenkoi was distributed in the Ili Region of Xinjiang; this record was based on two specimens not preserved in China (Eremchenko and Shcherbak 1986).A. a. alaicus was thought to be distributed near the south-western Xinjiang border close to Kyrgyzstan and Tajikistan, but no voucher specimens are available to support this distribution.Notably, historical records of A. alaicus in China have not been confirmed.
In 2022 and 2023, we observed skinks in Wuqia County, Kizilsu Kirgiz Autonomous Prefecture (KKAP) and Qapqal Xibe Autonomous County, Ili Kazakh Autonomous Prefecture (IKAP), Xinjiang, China, locations in which we had not previously observed them.We subsequently captured skinks from these two locations and conducted morphological and phylogenetic analyses.The results suggest that the skinks from KKAP belonged to A. eremchenkoi, a new record for this species in China.The skinks from IKAP were A. alaicus, confirming the distribution of A. alaicus in Xinjiang, China.In this study, we present the first known records of A. eremchenkoi and A. alaicus with voucher specimens from within China.In addition, we provide detailed descriptions of the morphology, phylogeny and natural history notes.
All specimens were euthanised and muscle or liver samples were dissected from the specimens, preserved in 95% ethanol and stored at -20 °C.All specimens were fixed in 10% buffered formalin and transferred to 75% ethanol for preservation.Liver and muscle tissues used for molecular analysis were preserved in 95% alcohol at -20 °C.All specimens were deposited at the Herpetological Museum, Xinjiang Agricultural University (XJAU), Urumuqi, Xinjiang, China.

Sequence extraction
Total DNA was extracted from tissue samples by using the Foregene Animal Tissue Genomic DNA Extraction Kit per the manufacturer's instructions.We referred to the primer sequences used by (Mirza et al. 2022).Additionally, we sequenced partial segments of the mitochondrial 16S rRNA (16S), 12S rRNA (12S) and NADH dehydrogenase subunit 2 (ND2) (Tables 1, 2).The PCR reaction volume was 25 μl, containing 1 μl template DNA, 1 μl each of upstream and downstream primers, 12.5 μl 2× Taq PCR Mix and 9.5 μl ddH 2 O.The PCR protocol used for amplification was as follows: 95 ℃ for 3 min, (denaturation temperature 95 ℃ for 30 s, annealing time ranged from 40 to 50 s, elongation temperature 72 ℃ for 1 min) × 36 cycles, 72 ℃ for 10 min, hold at 4 ℃ (Mirza et al. 2022).Gel electrophoresis was performed using 0.5% TBE solution and agarose to verify successful amplification of the samples.Subsequently, the successfully amplified PCR stock solution was sent to Sangon Biotechnology for purification and sequencing.

Molecular data and analyses
All sequences were aligned and manually edited using SeqMan in DNASTAR (Burland 1999).Sequences were aligned in Mega 7.0 (Kumar et al. 2016) using ClustalW (Thompson et al. 1994) with default settings.
Ultimately, a 12S sequence of approximately 356 bp, a 16S sequence of 389 bp, an ND2 sequence of 509 bp and a spliced sequence of 1323 bp were obtained.We collected reference sequences of the same genus from GenBank, with Scincella reevesii as the outgroup and constructed a phylogenetic tree of Ablepharus and its species within the genus by using Bayesian Inference (BI) and Maximum Likelihood (ML) to explore the affinities of the sample sequences in Ablepharus.The BI and ML analyses were performed using MrBayes and IQ-TREE in PhyloSuite (Zhang et al. 2020) with default parameters, respectively.The resulting phylogenetic tree was visualised using FigTree v.1.4.3 and the effective sample size (ESS) was assessed using Tracer 1.7.2 (Rambaut et al. 2018), with all ESS values for parameters > 200.Finally, the phylogenetic tree was visualised using ITOL v.6 (Letunic and Bork 2021).A posteriori probability (BPP) or ML bootstrap value (BS) > 95% was considered strong support for monophyly.
We used p-distance (uncorrected) in MEGE 7.0 (Kumar et al. 2016) to calculate the genetic distances of the three sequences: 12S, 16S and ND2.We divided the sample and reference sequences into groups by using MEGA and p-distance to calculate genetic distances amongst the three gene sequences.

Morphological data and analyses
Measurements of seventeen morphological characteristics, selected from published literature, were recorded to the nearest 0.1 mm using digital calipers from Jiang-miao Ran (Zhao et al. 1999;Qi et al. 2022).These characteristics were: snout-vent length (SVL), distance from tip of snout to vent; head length (HL), distance from the tip of the snout to the posterior border of the collar; head width (HW), distance across the widest point of the head; head depth (HD), highest point of the head; axilla-groin distance (AG), distance between posterior edge of forelimb insertion and anterior edge of hind-limb insertion; fore-limb length (FLL), distance from fore-limb insertion to the longest digit; hind-limb length (HLL), distance from hind-limb insertion to the longest digit; tail length (TL), distance between the cloaca and the tail top; eye diameter (ED); eye-narial distance (END), from anterior margin of eye to posterior margin of nares; internarial distance (IND), distance between the nares, supraocular count (SC); supralabial count; (SL), supralabial count before eyes; ventral count (VC), number of latitudinal scale columns from the midpoint of the fore-limb base to the cloaca; toe IV lamellae count (T4lam), number of enlarged, undivided lamellae beneath Toe IV; mid-body scale-row count (MBSR), number of longitudinal scale rows measured around the mid-body; and neck scale-row count (NSR), number of longitudinal scale rows measured around the neck.

Results
ML and BI phylogenetic trees were constructed, based on three mitochondrial genes (12S, 356 bp; 16S, 457 bp; ND2, 509 bp) from twenty species, with a total length of 1323 bp.The ML and BI analyses resulted in largely identical topologies (Fig. 2).
The uncorrected p-distance, based on 12S sequences, was up to 20%; based on 16S sequences, it was up to 20%; and, based on ND2 sequences, it was up to 27%.The uncorrected p-distance between Group KKAP and A. eremchenkoi was 1%/2%/2% in the 12S/16S/ND2 sequences, respectively and was less than 10% for A. alaicus 1, from Kyrgyzstan, 12S/16S/ND2: 4%/4%/9%.The uncorrected p-distance between Group IKAP and A. alaicus 2, from Kazakhstan, was 4% for the ND2 sequences.The results of the genetic distances (Suppl.material 2) were consistent with the results of the phylogenetic analyses (Fig. 2), where the Groups IKAP and KKAP were clustered into a branch with A. eremchenkoi and A. alaicus and the genetic distances of all four (Groups IKAP, Groups KKAP, A. eremchenkoi and A. alaicus) were lower than those of other species of the same genus, indicating the closeness of the relationship between them.All newly-collected specimens were largely similar to specimens of the original description of A. eremchenkoi and A. alaicus (Tables 3-5).Thus, we report the rediscovery of A. alaicus and specimens from the KKAP (A. eremchenkoi) as a new record in China.
Description of specimens from China.The sample size comprised 16 specimens, all collected by Lei Shi, Jing An and Tao Liang.The main description of this species is based on the male specimen (XND0808007; Figs 3, 4) whose tail had been naturally regenerated.Data and descriptions of the three female specimens (XND0808001, 002 and 005) are provided in parentheses in the following text (if different).The data available for the four voucher specimens are listed in Table 3.
Morphologies of the remaining specimens were similar to those four adult specimens; these data are in Suppl.material 3.
The recorded characteristics of the specimen were as follows: body was small, nearly uniform in thickness, with SVL 46.5 mm and mass 2.04 g and slender (BW/ SVL ratio 0.11) with an elongated trunk (AG/SVL ratio 0.53); imbricate scales were smooth and glossy; snout was slightly pointed; head was small and longer than it was wide (HL 9.6 mm, HW 6.6 mm, HD 4.9 mm); eyes were small; ED external ear opening was small with obviously projecting lobules; END was 3.4 mm; fore-limbs and hind-limbs were relatively short, the fore-limb was shorter than the hind-limb (FLL/HLL ratio 0.79) and the tips of the digits of the fore-limb and hind-limb met when the limbs were adpressed against each other along the body axis (except for XND20230808001); the tail was broken, but had regenerated and the regenerated tail was narrower than the body (4.6 mm cf.6.2 mm) and was shorter than SVL (35.3 mm cf.46.5 mm) despite tails generally being longer than SVL.The width of the rostral was greater than its height and it was in contact with the first supralabials, nasals,and fronto-nasal.Nostrils were circular and located at the centre of the nasal cavity.Frontal, fronto-nasal and a pair of prefrontals were connected to a point (seven of sixteen individuals); four of sixteen individuals' prefrontals were widely in contact with each other and frontal and frontal-nasal were separate from each other; three of sixteen individuals' frontal and frontal-nasasl were widely in contact with each other, prefrontals were separate from each other; two of sixteen individuals had three prefrontals, which made frontal and frontal-nasal separate from each other.Prefrontal fan-shaped, a pair of prefrontals were in contact with the postnasal, loreal and first supraocular.A large single frontal, irregularly wedge-shaped, was in broad contact with the third and fourth supraoculars and a pair of frontoparietal posterolaterally.Frontoparietal were widely in contact with parietal and interparietal scales and third and fourth supraoculars.The interparietal rhomboid was posteriorly in contact with parietals.Parietals were anteriorly in contact with frontoparietal, interparietal and fifth supraocular and were laterally touching   the upper posterior temporals.Three supraoculars and the eyes were surrounded by a circle of tiny irregular scales.There were four scales between the nasal cavity and eyes and one individual (XND20230808019) had five scales.
For seven supralabials, there was a tiny supraocular between the second and third scales (Figs 3, 4) on the right side (Fig. 4) and seven infralabials.The mental was wider than it was long and was in contact with the first infralabial laterally, postmental posteriorly.Postmental was large and single; four pairs of large chin-shields were present, with the first pair in contact and the second pair narrowly separated by a single medial scale.Dorsal scalation was homogeneous with four columns; longitudinal scale rows were at mid-body 26 (25-29 Colouration in life: Overall, in the one male, the dorsal was coppery brown; dark longitudinal spots were present on the edges of scales and generated three irregular black lines continuing on to the tail.White dots were grouped into six irregular lines along the back of them; the two external dots merged into light lines on the dorsal sides (Fig. 4).The lines on the dorsal sides began at the nasal base until the tail base and they were filled with rare light dots (Fig. 4).The bottom half of the dorsal side was white.The male abdomen was orange-red to the tail, but not the regenerated tail.Females and males were coloured similarly, but the abdomen was paler for females than males;  the outline of ventrals was black; and subadults and juveniles had abdomens similar to females, but without the orange-red colour.Activity, habitats and distribution.All 16 specimens were collected during the day: 16:00-18:00 h and 11:30-13:00 h.According to the residents, these regions receive snow from September to May; thus, the activity times ranged probably from May to August.These individuals were collected at the bottom of a hill, from under rocks and some individuals were collected from riverbeds, 40.20°N, 74.56°E, 3133 m elev., (observations from Yahui Huang).This species was observed in Wuqia County, China.Except for Yuqitashi, where we obtained the specimen, this species has been observed in Kalatashi, at 40.0559°N, 74.5941°E and 3004 m elev.and in Jigen Village, at 39.82°N, 74.1069°E and 2709 m elev., identified by images provided by Ya-hui Huang and Jin-Xin Gu, respectively.All individuals were located in a continuous valley (Fig. 1), with altitude ranging from 2709 m to 3133 m (Fig. 1).
Reproduction and diet.Viviparity.Of these individuals, three were gravid females, one female (XND0808001) laid four litters on the morning of 11 August 2023 and two females (XND0808002 and XND0808005) laid two litters on 23 and 24 August (Tables 4, 5) in the laboratory.On average, for these young, weight was 0.26 g, SVL was 22.9 mm and TL was 25.2 mm (Suppl.material 3).Juveniles were coloured and morphologically similar to adults, but had no orange-red colour on their abdomens.The diet of this species remains poorly understood, but they are thought to be carnivorous.
Description of specimens from China.The sample size comprised 14 specimens, all specimens were collected by Peng Ding, Lin Leng and Ke-fan Wu.The main descriptions of this species were based on one specimen (XND2023092704).Additional descriptions were based on the other 13 specimens (in parentheses).All specimen morphological data is in the Suppl.material 3.
The rostral was single; wider than it was high; and was in contact with the first supralabials, nasals and frontonasals.The nasal rhomboid comprised circular nostrils, located at the centre of the nasal cavity.Fronto-nasal was fan-shaped and connected to the prefrontals.Prefrontals were pentagonal, a pair of prefrontals were connected with a border between them, located between the fronto-nasal and frontal (seven of fourteen individuals); three of the 14 individuals had frontals, fronto-nasals and a pair of prefrontals connected by a point; 3 of the 14 individuals had the frontal and frontal-nasal widely in contact with each other and the prefrontals were separate from each other.The frontal was wedge-shaped, which contacts with the prefrontals, the third and fourth supraoculars and a pair of frontoparietals posterolaterally.Six of the 14 individuals had frontals in contact with fronto-nasals, the prefrontals were not in contact with each The plots were based on the male (XND0808007), the grey scale was the mutational scale between the second and third scales on the right side.
other.The second supraocular region, in contact with the frontal and prefrontal regions, was a single tiny supracular hexagon, between the second supraocular and prefrontal regions.A pair of frontoparietals were in broad contact with each other; besides each frontoparietal was in contact with the frontal, third, fourth supraoculars, the parietal and interparietal.The interparietal rhomboid, in contrast with the frontoparietals, was posteriorly in contact with the parietals.A pair of parietals contact each other; additionally, each parietal was in contact with the interparietal, frontoparietal, fourth supracular and temporals.There were three scales between the nasal cavity and eyes; 10 of the individuals had at least four scales on one side.There were seven supralabials, four loreals between the nasal and eyes and a fourth tiny loreal.The specimen had seven infralabials; three individuals had six infralabials on each side.The temporal 1+2 and the second subtemporal were large and trapezoidal.The mental was wider than long, in contact with the first infralabial laterally and postmental posteriorly.There was a single, large postmental with four pairs of large chin-shields; the first pair was in contact with the second pair narrowly separated by a single medial scale.Dorsal scalation was homogeneous with four columns; there were longitudinal scale rows at mid-body 25 (25-28).There were 28 (25-29) scales around the middle of the neck and 50 (42 -51) ventral scales.There were 17 toe IV lamellae.
Colouration in life.The back was coppery brown, with dark longitudinal spots on the edges of the scales, which generated three black lines continuing on-to the tail; there were white longitudinal spots in the middle of the scales, generating three irregular lines continuing to the back of the tail base.A dark sooty area on each side was sharply defined above, but faded below the belly.The dark sooty area began after the nasal cavity and ended at the middle of the tail (Fig. 5).The abdomen of the females was slightly orange-red during the breeding seasons.
Reproduction, activity, habitats, diet and distribution.Viviparity.All fourteen specimens were collected during the day, from 12:00 to 18:00 h; therefore, this species appears to be diurnal.These individuals were collected at the bottom of a hill at an altitude of 2466 m and the microhabitats were covered with shrubs and gravel.Their diet remains poorly understood, but they are thought to be carnivorous.This species has been observed in Qapqal Xibe Autonomous County, China and probably in adjacent Zhaosu County, which, along with Tianshan, has populations that are connected to those in Kazakhstan (Fig. 1).

Figure 1 .
Figure 1.Map showing the distribution of A. eremchenkoi and A. alaicus within China.The distribution map of A. alaicus was obtained from the IUCN Red List (Accessed on 17 September 2023).Photos by Lei Shi, Lin Leng and Tao Liang.

Figure 4 .
Figure 4. Schematic representation of head scalation of Ablepharus eremchenkoi.A. Lateral view; B. Dorsal view; C. Ventral view.The plots were based on the male (XND0808007), the grey scale was the mutational scale between the second and third scales on the right side.

Figure 5 .
Figure 5.The general aspect and close-up views of Ablepharus alaicus (XND2023092704) in life from Qapqal Xibe Autonomous County, Ili Kazakh Autonomous Prefecture, Xinjiang, China. A. Dorsolateral view of body; B. Ventral view of body; C. Dorsal view of head; D. Right side view of head; E. Ventral view of head.Photos by: Wei-Zhen Gao.

Table 1 .
Localities and GenBank accession numbers for all species used in this study.

Table 2 .
(Mirza et al. 2022mers used in the study for PCR amplification and sequencing(Mirza et al. 2022).

Table 3 .
Measurements (mm) and scale counts of adult Ablepharus eremchenkoi from Xinjiang, China.See Materials and Methods for abbreviations.* indicates dropped or regenerated tail.

Table 4 .
Descriptive statistics for female reproductive traits of Ablepharus eremchenkoi.

Table 5 .
Measurements (mm) and scale counts of juveniles of Ablepharus eremchenkoi.See Materials and Methods for abbreviations.