Phylogenetic analyses

The COI dataset subjected to phylogenetic analyses included 22 Scincella representatives from Cambodia and Vietnam and Sphenomorphus maculatus used as an outgroup to Scincella based on Pyron et al. (2013) (Table 2).

Nucleotide sequences were initially aligned using ClustalX 1.81 (Thompson et al., 1997) with default parameters, and then optimized manually in BioEdit 7.0.5.2 (Hall, 1999) and MEGA 7.0 (Kumar et al., 2016). The final alignment included 668 sites. Mean uncorrected genetic distances (P-distances) between sequences were determined with MEGA 7.0. MODELTEST v.3.06 (Posada & Crandall, 1998) was used to estimate the optimal model of DNA evolution. The best-fitting models selected for the COI dataset were SYM+I for the first, F81+I for the second, and HKY+G for the third codon positions, as suggested by the Akaike Information Criterion (AIC).

Phylogenetic trees were inferred using Bayesian inference (BI) and maximum likelihood (ML). BI was conducted in MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003); Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were run with one cold chain and three heated chains for four million generations and sampled every 1 000 generations. Five independent MCMCMC runs were performed and 1 000 trees were discarded as burn-in. We checked the convergence of the runs and that the effective sample sizes (ESS) were all above 200 by exploring the likelihood plots using TRACER v1.5 (Rambaut & Drummond, 2007). Confidence in tree topology was assessed by posterior probabilities (BPP) (Huelsenbeck & Ronquist, 2001). The ML analyses were conducted using Treefinder (Jobb et al., 2004). Confidence in tree topology was tested by non-parametric bootstrap analysis (MLBS) with 1 000 replicates (Felsenstein, 1985). We a priori regarded tree nodes with bootstrap (MLBS) values of 70% or greater and posterior probabilities (BPP) values over 0.95 as sufficiently resolved, those MLBS between 70% and 50% (BPP between 0.95 and 0.90) as tendencies, and those MLBS below 50% (BPP below 0.90) as unresolved (Felsenstein, 2004; Huelsenbeck & Hillis, 1993).

Systematics

The newly discovered population of Scincella from Mondulkiri Province represents an independent mtDNA lineage, with phylogenetic relationships to S. reevesii, S. rufocaudata, and S. rupicola (Figure 2). This population was clearly distinct in COI sequences from all examined congeners with P-distances in interspecific comparisons varying from 13.29% (with S. rufocaudata) to 19.92% (with S. melanosticta) (Table 3), indicating deep divergence in the examined mtDNA marker.

Morphologically the Mondulkiri population of Scincella also showed affinities with S. reevesii and S. rufocaudata; however, it can be easily diagnosed from these species and other congeners inhabiting the Indochina region by several morphological diagnostic characters (see Comparisons below). Herein, we describe this population as a new species.

Scincella nigrofasciata sp. nov.

Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8; Table 1, Table 2, Table 3, Table 4, Table 5 and Table 6.

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Figure 3

Female holotype of Scincella nigrofasciata sp. nov. (CBC02546) in preservative.

A: Dorsal view; B: Ventral view; C: Dorsal view of head; D: Lateral view of head; E: Ventral view of head. Scale bar: 5 mm. Photos by Thy Neang.

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Figure 4

Coloration in life of Scincella nigrofasciata sp. nov. (Photos by Thy Neang).

A: Female paratype (CBC02840); B: Male paratype (CBC02545).

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Figure 5

Morphology and coloration of fingers (A–D) and structure of hemipenes (E–H) of Cambodian Scincella species (Photos by Thy Neang).

A, E: Male paratype (CBC02545, SVL 50.2 mm) of Scincella nigrofasciata sp. nov.; B, F: Male (CBC02409, SVL 53.1 mm) of S. rupicola; C, G: Male (CBC1342, SVL 41.8 mm) of S. reevesii; D, H: Male (CBC01434, SVL 49.4 mm) of S. melanosticta.

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Figure 6

Differences in dorsal and dorsolateral views in life between Scincella species from Cambodia (Photos by Thy Neang).

A: Male of S. rupicola (CBC02409); B: Female of S. rupicola (CBC02339), both A and B from central part (Prey Lang) of Cambodia; C: Male of S. melanosticta (CBC01009); D: Male of S. reevesii (CBC02305); E: Female of S. reevesii (CBC01382); C, D, and E from Phnom Samkos Wildlife Sanctuary, Cardamom Mountains, southwest Cambodia.

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Figure 7

Dorsolateral views of representatives of Cambodian Scincella species (in preservative) (Photos by Thy Neang).

A: Male (CBC01454) of S. melanosticta; B: Male (CBC02409) and C: Female (CBC02339) of S. rupicola; D: Male paratype (CBC02545), E: Female paratype (CBC02840), and F: Female holotype (CBC02546) of Scincella nigrofasciata sp. nov.; G: Male (CBC1342) and H: Female (CBC01382) of S. reevesii.

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Figure 8

Male holotype of Sphenomorphus rufocaudatus (Darevsky & Nguyen, 1983) (ZISP 19797; now Scincella rufocaudata) (Photos by Nikolay A. Poyarkov).

A: Dorsal view; B: Ventral view; C: Dorsal view of head; D: Lateral view of head; E: Ventral view of head. Scale bar: 5 mm.

Holotype: CBC02546, adult female, collected by Thy Neang on 25 September 2016 at N12${}^{\circ }$19${}^{\prime }$12.3${}^{″}$, E107${}^{\circ }$04${}^{\prime }$20.8${}^{″}$, 508 m a.s.l in Keo Seima Wildlife Sanctuary, O’Raing District, Mondulkiri Province, Cambodia.

Paratypes: CBC02545, adult male, CBC02840, adult female, and CBC02841–42, two subadults, collected by Thy Neang at the same date and locality as given for the holotype.

Referred materials: CBC02843–45, three juveniles, collected by Thy Neang at the same date and locality as given for the holotype.

Diagnosis: The new species was assigned to the genus Scincella Mittleman, 1950 as it shows morphometric and meristic characters matching the diagnosis for this genus. Scincella nigrofasciata sp. nov. can be diagnosed from other congeners by the following combination of morphological attributes: (1) slender and medium-sized, SVL 40.0–52.6 mm; (2) tail relatively long, TaL/SVL (1.25–1.94); (3) FIL/SVL 0.20–0.22; (4) HIL/SVL 0.30–0.33; (5) forelimbs and hind limbs either slightly overlapping (0.4–2.2 mm) or slightly separated (1.9–2.3 mm) when adpressed to body toward each other; (6) infralabials 6; (7) supraciliaries 7–8; (8) prefrontals in broad contact; (9) primary temporals 2; (10) nuchal scales weakly enlarged; (11) external ear opening without lobules; (12) dorsal scales smooth: MBSR 32–33, PRVSR 69–74, VS 65–69, DBR 8; (13) SDLT4 15–17; (14) coloration pattern with dorsum dark brown/greyish-brown in life with 5–7 regular discontinuous dorsal dark stripes (formed by series of dark dots or elongated black spots), including paravertebral stripes, wide black dorsolateral stripes, 2–3 scale rows in width, starting from posterior corner of eye and continuing to lateral side of tail, extending 52%–86% of total tail length; and (15) hemipenis bifurcating about 63% of its total length to base.

Description of holotype (Figure 3): A gravid adult female, SVL 52.6 mm; tail relatively long, TaL 84 mm, (TaL/SVL 1.6); head elongated, HL 8.5 mm (HL/SVL 0.16), longer than wide, HW 6.1 mm (HW/HL 0.72), slightly depressed, HD 4.2 mm (HD/HL 0.49). Neck rather slender, slightly distinct from head.

Head: Snout rounded in profile and dorsal view, SnL 3.4 mm, more than twice as long as TD (1.5 mm); STL 8.8 mm; SFlL 16.9 mm, comprising about one third of SVL; ear vertically oval, TD 1.5 mm; ED 2.5 mm; diameter of cornea 1.3 mm; rostral broad, (width 1.7 mm), almost three times greater than height (0.6 mm), visible from above, in contact with 1^st SL laterally, nasals and frontonasal posteriorly; supranasals absent; frontonasal broad, subtrapezoidal in shape, anterior side forming almost straight suture (0.6 mm) with rostral, posterior width 1.7 mm, as wide as rostral, little more than twice as wide as length (0.8 mm), in contact with nasals and 1^st loreal laterally, posterior margin slightly overlapping prefrontals; prefrontals in broad contact, laterally bordered by two loreals, frontal posteriorly; frontal elongated (length 2.8 mm), kite-shaped, posterior part much longer than anterior; greatest width anteriorly 1.4 mm, twice as narrow as length (width/length 0.5); frontal in contact with 1^st and 2^nd supraoculars laterally, frontoparietals posteriorly, anterior corner of rostral end slightly separating posterior portions of prefrontals medially, posterior corner of frontal slightly overlapping medial suture between frontoparietals; frontoparietals two, each diamond-shaped, together forming a butterfly-shape with median suture 1.2 mm, in contact with 2^nd, 3^rd, and 4^th supraoculars laterally, interparietal and parietals posteriorly; interparietal rather small, kite-shaped, with posterior portion little longer than anterior, in contact with parietals posteriorly, anterior corner of interparietal acute, slightly intruding into median suture between frontoparietals; parietals large, in contact with each other posteriorly (suture 0.6 mm behind posterior corner of interparietal), narrowly contacting 4^th supraocular and posterior supraciliary scale, in broad contact with upper secondary temporal laterally and four nuchal scales posteriorly. Naris rounded, laterally pierced in nasal scale; nasals in contact with 1^st SL ventrally, frontonasal dorsally, 1^st loreal posteriorly; loreals two, anterior loreal rhomboidal, in contact with 2^nd SL ventrally, frontonasal and prefrontal dorsally, posterior loreal subtrapezoidal, in contact with 2^nd and 3^rd SL ventrally, preocular and upper presubocular posteriorly, prefrontal and anterior supraciliary scale dorsally; preocular one, elongate, triangular, in contact with anterior supraciliary scale dorsally, anterior edge of orbit posteriorly, anterior presubocular ventrally; supraciliaries eight, anterior two largest; supraoculars four, first two contacting frontal, second to third contacting frontoparietal; presuboculars three, posterior-most slightly intruding into suture between 3^rd and 4^th SL, anterior-most triangular, slightly larger than posterior-most presubocular; suboculars two, both contacting 4^th SL, anterior slightly overlapping posterior one, slightly overlapped by lower presubocular, posterior broadly overlapping lower postsubocular, both bordered above by granular scales of lower eyelid; postoculars two; postsuboculars four (left side) and five (right side), lowest slightly intruding between 4^th and 5^th SL, uppermost largest; lower eyelid with distinct transparent disc (window) bordered above by small palpebral scales; supralabials six, 1^st smallest, 4^th located ventral to window of eye, 5^th largest; infralabials six, 1^st smallest, 5^th largest; primary temporals two, lower larger, sub-rhomboid, anteriorly in contact with 3^rd and 4^th postsuboculars, ventrally with 5^th and 6^th SL, posteriorly with lower secondary temporal, upper primary temporal subrhomboid, anteriorly in contact with 1^st and 2^nd postsubocular anteriorly, posteriorly with both secondary temporals; secondary temporals two, lower smaller, overlapping upper, in contact with 6^th SL ventrally, upper secondary temporal about twice as large as lower, in contact with posterior-most postsubocular anteriorly, with parietal dorsally and nuchal scale posteriorly; nuchal scales four, bordering posterior edge of parietals, slightly enlarged in comparison with adjacent posterior scales. Mental rounded, width (1.6 mm) more than twice as wide than long (0.7 mm), in contact with 1^st IL laterally, postmental posteriorly; postmental large, width (2.0 mm) greater than length (1.1 mm), in contact with 1^st and 2^nd IL laterally, 1^st chinshield posteriorly; chinshields in three pairs, 1^st pair in broad contact median with each other, contacting 3^rd IL laterally, 2^nd pair separated by subtriangular gular scale, contacting 4^th IL laterally, 3^rd pair separated medially by three gular scales, in contact with 5^th and 6^th IL laterally and three gular scales posteriorly.

Body, limbs, and tail: Body scales smooth, cycloid, imbricate; dorsal scales between dorsal stripes ½+8+½, same size as ventral scales, slightly larger than those on body sides and gular scales; scales on anterior flanks between tympanic region and posterior margin of axilla smaller than adjacent dorsal scales; MBSR 32; PRVSR 74; VS 69; enlarged preanal scales two, median scales overlapping outer; subcaudal scales 111, anterior in three rows, reducing to two at quarter of tail length and one row about half way to tail tip, slightly larger than surrounding scales. Trunk relatively long, TrunkL 29.7 mm, little more than half of SVL, more than addition of FIL and HIL, ratio of TrunkL/(FIL+HIL) 1.1; forelimb short, FIL 10.8 mm (FIL/SVL 0.21); forearm short, rather slender, FoL 7.4 mm (FoL/SVL 0.14); hindlimb longer than forelimb, HIL 16.6 mm (HIL/SVL 0.32), limbs separated by 2.3 mm when adpressed (4.4% of SVL); digits slender; SDLF4 11; SDLT4 16.

Coloration in life: In life, the female holotype CBC02546 had the same color as female paratype CBC02840 (Figure 4). Dorsal surface of head, dorsum, and base of tail dark bronze-brown, side of head between tip of snout and forelimb insertion dark brown; dorsal surface of remaining tail reddish-brown. Dark broken regular dorsal stripes anteriorly and on tail (5) and posteriorly on body (7), formed by series of dark dots or elongated black spots, including wider dark paravertebral stripe; anterior part of dorsum with dorsal stripes formed by series of dots, posterior part of dorsum with dark dot-formed regular dorsal stripes reaching base of tail, continuing with dark stripes on dorsal surface of tail, extending about one-third of tail length; light laterodorsal stripes from behind eye, through temporals, along dorsolateral scale row to lateral sides of tail, and fading at one-third of tail length; large distinct regular black longitudinal dorsolateral stripe on each side of body, covering two to three scale rows, starting as narrow stripe covering about one scale row, running from posterior corner of eye through upper temporals, above tympanum, expanding wider to two scale rows above axilla, running below light dorsolateral stripe, along upper flanks through upper angle of groin to lateral surface of tail, becoming indistinct at posterior lateral tail (~12 mm from tail tip); body flanks ventrally with whitish-beige longitudinal streaks and dark markings on bluish-brown background; ventrolateral surfaces from below level of eye to axillary region with longitudinal whitish-grey streaks and dark marking on reddish-brown background; lateral surfaces of tail reddish-brown; dorsal surfaces of limbs with irregular dark blotches on dark brown background. Ventral surfaces of head, gular region, body, and limbs uniformly white; ventral surface of tail uniform pinkish-cream. Palmar surfaces of hands and thenar surfaces of feet dark grey. Iris light-grey.

Coloration in preservative: In preservative, dorsal surfaces of holotype turned dark greyish-brown; wide dorsolateral black stripe remained distinct; head with faint irregular dark spots, pineal ocellus present as single white dot on posterior part of interparietal; sides of head and supralabials with dark mottling; infralabials with dark spots; lateral sides of tail with small dark spots; throat and ventral surface of body and limbs greyish-cream; ventral surface of tail lighter cream; palmar surface of hand, fingers, and toes dark grey (Figure 3, Figure 7D, E, F).

Variation: Paratypes (Table 1) resemble holotype in most morphometric and meristic characters and coloration. Noteworthy variation is that the holotype has six SL on each side of the head, whereas all paratypes have seven. CBC02545 has two supraoculars on left side, second posterior one larger with clear short suture indicating incomplete fusion of two posterior supraoculars; three supraoculars on right side, third with clear short suture indicating incomplete fusion with fourth posterior-most supraocular; one distinct pair of enlarged nuchal scales (remaining specimens have weakly enlarged nuchals) and first pair of chinshields in narrow contact with each other (vs. in broad contact in other specimens). CBC02545 has four postsuboculars and CBC02840 has five postsuboculars on both sides. Male paratype exhibits greater tail length (97.3 mm) than female holotype and paratypes. All specimens have six nuchal scales posterior to parietals, except subadult CBC02842, which has seven. In life, male shows distinctly more reddish-brown coloration of dorsum, with indistinct mid-dorsal stripe; throat, lower body flanks, ventral surfaces of body, lateral and ventral surfaces of tail in male show distinct reddish-orange coloration (Figure 4B). In preservative, reddish-orange coloration faded and became paler (Figure 7D).

Natural history: The species was recorded from semi-deciduous lowland forests at elevations ca. 400–500 m a.s.l. in Keo Seima Wildlife Sanctuary from the eastern plain of Cambodia. Most specimens were encountered during the day, but juveniles CBC02843–45 were encountered at night among leaf litter. The holotype female CBC02546, paratype male CBC02545, and subadult CBC02841 were spotted moving near rotten logs, female CBC02840 was moving along the ground on the forest floor, and subadult CBC02842 was found under a rotten log. Diet and reproductive biology of the new species remain unknown. The gravid female holotype carried two eggs.

Etymology: The specific epithet is from the Latin words “niger” for “black” and “fascia” for “band”, in reference to the wide black dorsolateral stripes typical for this species.

Distribution: To date known only from the type locality in Keo Seima Wildlife Sanctuary, O’Raing District, Mondulkiri Province, Cambodia at elevations ca. 400–500 m a.s.l.. However, the discovery of this species in adjacent areas of southern Vietnam is highly expected.

Comparisons: The morphological characters distinguishing the new species from its Southeast Asian congeners are summarized in Table 4. Morphological comparisons of Scincella species found in Cambodia are given in Table 5. Scincella nigrofasciata sp. nov. can be diagnosed from S. apraefrontalis Nguyen, Nguyen, Böhme and Ziegler, 2010 of Vietnam by its longer SVL (40.0–52.6 vs. 36.1 mm), greater number of IL (6 vs. 5), DBR (8 vs. 4), MBSR (32–33 vs. 18), PRVSR (69–74 vs. 52), and VS (65–69 vs. 50), and prefrontals in broad contact (vs. prefrontals absent); from S. monticola (Schmidt, 1925) of Vietnam and China by having a longer SVL (40.0–52.6 vs. 31.8 mm), two primary temporals (vs. one), fewer EnLN (0–1 vs. 3–4), and greater number of DBR (8 vs. 4), MBSR (32 vs. 22–26), PRVSR (69–74 vs. 52–59), VS (65–69 vs. 52–58), and SDLT4 (15–17 vs. 10–13); and from S. punctatolineata (Boulenger, 1893) of Thailand and Myanmar by longer SVL (40.0–52.6 mm for three adults and single subadult specimen, SVL 50.2–52.6 mm for three adults vs. 37.6–40.2 mm), greater number of MBSR (32–33 vs. 22–28), two primary temporals (vs. one), and greater number SDLT4 (15–17 vs. 13–15). Scincella nigrofasciata sp. nov. can be distinguished from S. darevskii Nguyen, Ananjeva, Orlov, Rybaltovsky and Böhme, 2010 of Vietnam by having a much shorter SVL (40.0–52.6 vs. 88.6 mm), fewer supraoculars (2–4 vs. 5), two primary temporals (vs.one), and greater number of DBR (8 vs. 6), MBSR (32–33 vs. 28), and PRVSR (69–74 vs. 62); from S. doriae (Boulenger, 1887a) of Myanmar and China by having a shorter SVL (40.0–52.6 vs. 58.6 mm), fewer EnLN (0–1 vs. 3–5), slightly fewer VS (65–69 vs. 70–79), 5–7 discontinuous regular dark dorsal stripes (vs. dorsum caramel brown with small brown spots), and distinct wide black dorsolateral stripes (vs. dark brown dorsolateral stripes broken up by whitish spots); from S. rara Darevsky & Orlov, 1997 of central Vietnam by having fewer EnLN (0–1 vs. 3), greater number of MBSR (32–33 vs. 24) and PRVSR (69–74 vs. 53), and single row of basal subdigital pads (vs. double row of basal subdigital pads); from S. victoriana (Shreve, 1940) of Myanmar by having a shorter SVL (40.0–52.6 vs. 57.5 mm), fewer EnLN (0–1 vs. 3), smooth dorsal scales (vs. keeled), and a greater number of PRVSR (69–74 vs. 50–54) and VS (65–69 vs. 53–56). The new species can be distinguished from S. ochracea (Bourret, 1937) of Vietnam and Laos by its longer SVL in males (50.2 mm, n=1 vs. 34.2–45.4 mm, n=6), lack of lobules around external ear opening (vs. 2–4 lobules), dark brown dorsum with 5–7 discontinuous regular dark dorsal stripes (vs. silver-grey with a dark vertebral stripe), and distinct wide black dorsolateral stripes (vs. dark brown flanks broken up by light spots).

Among the Cambodian species, Scincella nigrofasciata sp. nov. can be distinguished from S. melanosticta (Boulenger, 1887b) of Cambodia, Myanmar, Thailand, and Vietnam by its comparatively shorter forelimbs (FIL/SVL 0.20–0.22 vs. 0.23–0.27), comparatively shorter hind limbs (HIL/SVL 0.30–0.33 vs. 0.35–0.37), adpressed limbs overlapping 0.4–2.2 mm in males and subadult specimens and separated by a 1.9–2.3 mm gap in adult females (vs. adpressed limbs widely overlapping 4.5–8.2 mm), two primary temporals (vs. one), fewer DBR (8 vs. 10), and 5–7 discontinuous regular dark dorsal stripes (vs. dark brown dorsum with dark dense spots without obvious striped pattern). The new species can be distinguished from S. rupicola by its comparatively shorter hind limbs (HIL/SVL 0.30–0.33 vs. 0.36–0.40), adpressed limbs overlapping 0.4–2.2 mm in male and subadult specimens and separated by a 1.9–2.3 mm gap in adult females (vs. adpressed limbs overlapping 3.5–7.2 mm), fewer SDLT4 (15–17 vs. 18–21), fully everted hemipenis bifurcating at 63% of total hemipenis length, n=1, Figure 5E (vs. 69%–77%, n=3, Figure 5F), comparatively more slender fingers and toes (Figure 5A vs. Figure 5B), 5–7 discontinuous regular dark dorsal stripes, Figure 4, Figure 7D–F (vs. dark blotches on dorsum in females and uniform reddish brown pattern without dark markings in males in S. rupicola; Figure 6A–B, Figure 7B–C).

In both morphometric and meristic characters Scincella nigrofasciata sp. nov. is most similar to S. reevesii and S. rufocaudata. However, the new species can be distinguished from S. reevesii by slightly shorter forelimbs (FIL/SVL 0.20–0.22 vs. 0.24–0.30), generally shorter hind limbs (HIL/SVL 0.30–0.33 vs. 0.34–0.43), comparatively shorter forearms (FoL/SVL 0.14–0.16 vs. 0.17–0.19, Table 5), adpressed limbs overlapping 0.4–2.2 mm in males and subadult specimens and separated by a distance of 1.9–2.3 mm in females (vs. overlapping 3.9–6.5 mm in both sexes), 5–7 dark discontinuous regular dark dorsal stripes (vs. irregular dark vertebral line and dark dorsal spots), wide distinct black dorsolateral stripes, continuing to lateral sides of tail, Figure 4 (vs. dark dorsolateral stripes less distinct and broken up by light spots and only extending to tail base in both sexes, Figure 6E, D), comparatively more slender fingers and toes (Figure 5A vs. Figure 5C), and dark brown palmar surfaces of hands and lower surface of fingers and toes, Figure 5A (vs. light grey palmar surfaces of hands, fingers and toes, Figure 5C).

Scincella nigrofasciata sp. nov. can be distinguished from S. rufocaudata by prefrontals in broad contact (vs. prefrontals separated), comparatively shorter hind limbs, (HIL/SVL 0.30–0.33 vs. 0.37), fewer IL (6 vs. 7), fewer DBR (8 vs. 10, Table 4, Table 5, Table 6), 5–7 discontinuous regular dark dorsal stripes (vs. 1–3 dark stripes with spots, Figure 8), and distinct wide black dorsolateral stripes continuing along tail (vs. black stripes broken, ending at tail base, Figure 4) (Table 5).