﻿A new species of the Cyrtodactylusquadrivirgatus complex (Chordata, Reptilia, Squamata, Gekkonidae) from Sumatra Barat, Indonesia

﻿Abstract Among the six species of Cyrtodactylus occurring in Sumatra, two species were described based on non-Sumatran type series, C.consobrinus and C.quadrivirgatus. The latter species was described originally from Thailand thus the wider distribution in Sumatra should be clarified taxonomically. Cyrtodactylusquadrivirgatus from Sumatra Barat was examined using both morphology and the Natrium Dehydrogenase Subunit 2 (ND2) gene to clarify its taxonomic status and phylogenetic placement. It was found that these specimens form a sister clade to all other species of the sworderi group from Peninsular Malaysia and the genetic distance ranges from 20–24.3%. This subset is herein described as a new species. The new species is readily distinguished from C.quadrivirgatus and other Sumatran species by a combination of characters: small size SVL 37.5–53.78 mm; longitudinal rows of dorsal tubercles 16–19; paravertebral tubercles 31–41; ventral scales 32–43; 24–49 enlarged precloacal and femoral scales; precloacal pores rarely present; no precloacal depression; two postcloacal tubercles on each side; 14–19 subdigital lamellae on forth toe; 9–15 supralabial scales; 9–12 infralabial scales; three or four internasal scales; and 3–6 gular scales that border the first pair of postmental scales. This work underscores the importance of clarifying widely distributed species for taxonomic validation.


Introduction
Cyrtodactylus quadrivirgatus Taylor, 1962 was originally described from Khao Chong Forest Experiment Station, Trang Province, Thailand. It ranges from southern Thailand, Peninsular Malaysia and adjacent islands, Singapore to northern Sumatra (Grismer 2011) and Mentawai islands (Teynie et al. 2010), from sea level to 1400 m above sea level (Johnson et al. 2012).
Along Peninsular Malaysia, the populations of C. quadrivirgatus exhibit coloration differences among different localities. The south population has four dark dorsal stripes, the upland population has two dorsolateral stripes and medial blotches, and the other populations possess only blotches instead of stripes. Although there was obvious variation among populations, the ND2 p-distance showed that they were separated from each other by 3.3%-5.8% (Johnson et al. 2012).
Meanwhile, the population from Sumatra was not examined either morphologically or molecularly, leaving this population unknown in term of its taxonomic status and phylogenetic placement. We began surveying Cyrtodactylus Gray, 1827 in Sumatra Barat Province in 2020 and found them from lower elevations, approximately 8 m a.s.l. to 712 m a.s.l. Through careful examination, we wanted to establish whether C. quadrivirgatus from Sumatra Barat should be treated as a distinct species and into which lineage it fell.

Morphological analysis
Color notes were observed from digital images of living individuals prior to preservation. If in case the individual displayed stress coloration, we placed them in the cage mimicking the natural habitat and waited until the natural coloration appeared. The individuals under the stress condition showed black coloration along their dorsum, causing the disappearance of the black stripes and bands on the dorsum.
The following measurements were taken with a dial caliper to the nearest 0.5 mm following Hartmann et al. (2016) and Johnson et al. (2012):

SVL
Snout-vent length, measured from the tip of snout to the vent; AX Axial length, measured from the posterior margin of the forelimb at its insertion point on the body to the anterior margin of the hind limb at its insertion point on the body; TL Tail length, measured from the vent to the tip of the tail, original or regenerated; AL Arm length, measured insertion of antebrachium with body wall to claw of longest finger; To make clear the counting of scales (supralabials and infralabials, precloaco-femoral scales) and detecting the presence of pores, we used a staining technique with methylene blue in 70% alcohol (Harvey et al. 2015). We determined male specimens by the enlarge hemipenial pockets and then confirmed the identification by making a small incision laterally at the base of the tail (Riyanto et al. 2021).

Laboratory protocols
Total genomic DNA was extracted from the livers using the Qiagen DNeasy tissue kit (Valencia, CA, USA) following the standard protocol for animal tissue. The amplification of the Natrium Dehydrogenase Subunit 2 (ND2) gene and partial flanking tRNAs was done by using Polymerase Chain Reaction (PCR) under the following condition: 2 min at 95 °C followed by 33 cycles of 95 °C for 35 s, annealing at 54 °C for 35 s, extension at 72 °C for 35 s and a final extension step of 10 min at 72 °C. Amplifications were carried out in 25-µl volume vials consisting of 2.5 µl genomic DNA (concentration: approximately 100 ng), 0.4 µм each primer and 1× GoTaq Green Master Mix (Promega, Wisconsin, USA). The primers used in this study followed Macey et al. (1999a): L4437b (5'-AAGCAGTTGGGCCCATACC-3') and L5002 (5'-AACCAAACCCAACTAC-GAAAAAT-3'). The PCR product was then sent to the sequencing service 1 st BASE (https://base-asia.com/) through Genetika Science Indonesia Limited Liability Company. The previous two primers were also used for sequencing.

Phylogenetic reconstruction
Sequences were uploaded, assembled, and edited in Geneious Prime 2022.2.2 (http://www.geneious.com/). All sequences, ingroup and outgroup (Table 1), were aligned using CLUSTALW implemented in CIPRES Science Gateway. The fasta output of alignment was used for RAxML and uncorrected p-distance calculation. We reconstructed phylogenetic relationships using maximum likelihood analysis that was performed using RAxML HPC Black Box (1000 bootstrap replicates) implemented in CIPRES Science Gateway portal (Miller et al. 2010; accessed through https://www.phylo.org/). Nodal support with bootstrap value ≥ 70 was considered as significantly supported (Hillis and Bull 1993). The tree resulted from RAxML was visualised and edited in iTOL v. 6 (Letunic and Bork 2021; available at https://itol.embl.de/) and in Photoshop C6 64-bit. We also calculated uncorrected p-distances using MEGA 7 with delete option for the gaps (Kumar et al. 2016).

Description (and variation).
Small-sized Cyrtodactylus with SVL of 37.5-53.78 mm; the length of the tail is 31.4-54.77 mm including the original or regenerated tip; the axial body length is 16.65-24.31 mm (Fig. 3). The head is triangular in dorsal view with moderate length (HL/SVL= 0.25-0.31), wide (HW/HL 0.52-0.63), and slightly flattened (HH/HL= 0.29-0.45), distinguishable from neck; medium length of snout (SL/HL 0.33-0.45) and rounded; snout longer than eye diameter (SL/OD 1.38-2.03); eyes large (OD/HL 0.18-0.27); ear openings oval and small (EL/HL 0.02-0.1); eye to ear distance greater than diameter of eye (OEL/OD 0.89-1.54); postorbital and around ear region consists of enlarged tubercles; scales on post nasal to preorbital and post-rostral to frontal region slightly larger in size than scales on the parietal part and occiput; region of parietal containing small scales intermixed with weak, scattered, rounded tubercles while occiput region contained slightly enlarged tubercles (Fig. 4). The nares are oval, bordered by rostral anteriorly, by supranasals and internasals dorsally, by 1 st supralabial ventrally. Supranasal scales larger than post-nasal scales. The supranasal scales as large as intersupranasals and separated from each another by three or four intersupranasal scales (Fig. 4F).
The triangular mental is bordered laterally by first infralabial and posteriorly by right and left first postmental. First postmentals medially connected each other for ~ 30% of their length. Second postmentals in contact with 1 st and 2 nd infralabials (N = 2) (Fig. 5A), separated from infralabials by relatively smaller scales (N = 1) (Fig. 5B), by relatively similar-sized scales (N = 5) (Fig. 5C), and by relatively larger scales (N = 1) (Fig. 5D). Right scale contacts with 1 st and 2 nd infralabials but the left only with 2 nd infralabial (N = 2) (Fig. 5E), or the right contacted with 2 nd infralabial and the left with small part of 1 st infralabial and large part of 2 nd infralabial (Fig. 5F). Right and left second postmentals are bordered by 3-6 relatively smaller scales (Fig. 5).
Body moderate in length (AX/SVL 0.38-0.53); defined ventrolateral fold with tubercles smaller than dorsal tubercles; dorsum with small scales interspersed with large conical or pyramidal, tubercles most dense on flanks; tubercles extending from occipital region to the base of tail, tubercles on tail largest; 16-19 tubercles between lateral fold in middle of body; 31-41 tubercles of paravertebral from posterior insertion of arm to body to anterior of femur insertion to body; 32-43 ventral scales larger than dorsal scales; ventral scales in middle part slightly larger than those near the ventrolateral folds; from middle of body, scales are smaller anteriorly to the head, ventrum, and posteriorly until groin region (Fig. 6). Forelimbs medium length (AL/SVL 0.33-0.4); granular scales on upper arm larger than those on dorsum of body (~ 2-3 ×larger); without tubercles; lower arm with smaller scales than upper arm scales, intermixed with weak tubercles slightly larger than weak tubercles on parietal parts; hindlimbs also moderate in size (LL/SVL 0.40-0.52); more robust than forelimbs; covered dorsally by granular scales intermixed with large, rounded tubercles; ventral scales of thigh larger than dorsals; 14-19 subdigital lamellae on 4 th toe. Continuous enlarged precloacal and femoral scales present (N = 24-49); no specimen has precloacal groove/ depression; enlarged post-precloacal scales present; two post-cloaca tubercles on left and right base of tail, mostly connected to each another (Fig. 7).
Tail length ~ 1.1 × of SVL, circular in cross-section but tapering at the end portion; tubercles on base of tail dorsally similar in size to those on body dorsum; 4-11 black dorsoventral stripes separated by white stripes; black stripes on venter more faded than on dorsal; part; no median, transversely enlarged, series of scales on the subcaudal; subcaudal cycloidal scales relatively larger than dorsal (Fig. 7).
Coloration in life. Ground color of body dorsum dark grey to brown; top of head blackish with irregular broken spots scattered on parietal region to nostril; on occipital regions three short black lines extending longitudinally: one in the middle, two begin behind each side of eyes almost parallel to the supraorbital regions; those three short black bands stop at approximately parallel to ears, after which there is a transverse white line extending from each pre-ear region; after the white line, there are two black lines at the nape of the neck that extend backwards, then some meet at an angle and some remain separate, as if these two lines continue the black line originating from the back of the eye parallel to the supraorbital area; after the meeting, there are two lines that separate to the back of the tail, and some are still united to the tail so that it tends to look like a black transverse band; in individuals with the two midlines converging, the confluence of the two lines begins just before the anterior part of the upper arm; there are eight or nine rows of black transverse bands that are counted from the beginning of the union of the two lines to the base of the tail; on the dorsolateral, there are two black lines that extend from behind the eyes to the base of the tail; unpatterned black blotches or obscure irregular black banding on limbs; black and white bands on tails; the width of the black line increases towards the posterior; and the white is opposite; in some individuals, the above-mentioned black stripes are not clear and not strong along the dorsal and dorsolateral body. Ventral surface of head, trunk, and limbs are white, pale grey to cream; ventral surface of tail cream in the first third at the anterior, then the rest to the posterior tends to black with narrow white rings (Fig. 8).
Coloration in preservative. Ground color of dorsal trunk, limbs, and tail brown to dark; parietal part to the tip of snout paler than any other parts of dorsum; the individuals with unclear or weak black lines on middle dorsum and dorsolateral tend to be dark from the nape to the base of the tails; black lines on nape and trunk still visible; tail with black and white bands; ventral head, trunk, limbs whitish to dark brown. Fresh specimens darker than the others both in ventral or dorsal parts of the body (Fig. 3).
Habitat. We collected the type series in the primary forest of LANR, SG, and SS with elevation ~ 380-767 m a.s.l. and we encountered non-vouchered indi- Figure 7. Tail in dorsal A and ventral B views, precloacal and femoral view (C) and ventral of 4 th toe (D). All images from holotype specimen (UNP070). Images not to scale. viduals from ~ 7 m a.s.l. At SG, this species was found on leaves measuring ~ 7-10 cm width and on twigs, ~ 1 m above the ground, 1-3 m from the edge of the rocky stream. The stream that empties into the waterfall has a breadth of ~ 2 m with a heavy flow. Fewer specimens were found closer to the waterfall. At LANR and SS, this species occupied the same microhabitat as the SG population, but the stream at this location is wider (~ 5-7 m width; Fig. 9). We also encountered this species (an unvouchered individual) at a lower elevation of 7 m a.s.l. in Bungus Selatan village. At this location, the gecko was perching on a bush leaf just beside the paddy field at ~ 70 cm above the ground. Another unvouchered individual was in the Sarasah Uwak waterfall area but far from the waterfall, perching on bushes at ~ 60 cm above the ground (Fig. 10).
Distribution. Currently, this new species is found only in Sumatra.   Etymology. The specific epithet awalriyantoi is in reference to the Indonesian herpetologist, Awal Riyanto. He has dedicated much of his time researching Indonesian Cyrtodactylus from Indonesia, as well as patiently and continuously supervising many younger amphibian and reptile taxonomists from both academic institutions and independent positions. Moreover, his contribution to the study of amphibians and other reptiles is significant for Indonesian herpetological knowledge and conservation.

Discussion
Previously, the sworderi group of Cyrtodactylus contained five species of which four are endemic to Peninsular Malaysia: living in lowland swampy habitats (C. sworderi; Taylor 1962), upland habitats (C. tebuensis; Grismer et al. 2013), and in karstic habitats (C. guakanthanensis and C. gunungsenyumensis; Grismer et al. 2014. The fifth species, C. quadrivirgatus, is a habitat generalist that is widely distributed from Thailand to Sumatra (Grismer 2011). Our study revealed that C. quadrivirgatus from Sumatra Barat differs from the Peninsular Malaysian population based on molecular and morphological evidence. With this addition, Sumatra currently supports six species of Cyrtodactylus in total, but the number of species endemic to this mainland is five: C. agamensis, C. lateralis, C. psarops, C. semicinctus and C. awalriyantoi.
Widely distributed species in Cyrtodactylus are most likely questionable, for example, two potentially new species have been detected within the C. marmoratus complex from southern Sumatra (O'Connell et al. 2019). This study also showed that widely distributed species like C. quadrivirgatus need confirmation. At the current state of knowledge, only C. consobrinus has a wide distribution, originally described from Sarawak (Borneo) and reported from Sumatra (Teynie et al. 2010). Like the new species C. awalriyantoi, its presence in Sumatra most likely warrants taxonomic validation.