Sex was determined by inspection of the presence or absence of hemipenes. Measurements were taken to the nearest mm with digital calipers. Paired meristic characters are given as left/right. The methodology of measurements and meristic counts followed Teynié et al. (2015) and Ziegler et al. (2018). The following characters and ratios were evaluated and calculated (Table 1): snout-vent length measured from tip of snout to anterior margin of cloaca (SVL); tail length measured from posterior margin of cloaca to tip of tail (TaL); total length, corresponding to SVL + TaL (TL); ratio of tail length to total length (TaL/TL); number of maxillary teeth, counted by investigating the right maxilla for teeth/sockets. The pholidosis characters taken or counted are as follows: dorsal scale rows counted at one head length behind head, at midbody, and at one head length before vent, respectively. The number of ventrals, subcaudals, supralabials, infralabials, suboculars, loreals, preoculars, postoculars, temporals and cloacal scales were counted. Morphological comparisons were based on data from Teynié et al. (2015), Nguyen et al. (2015), Teynié and Hauser (2017), Ziegler et al. (2018), and Cai et al. (2020).

Museum abbreviations are as follows:

ZISP Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia;

IEBR Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hanoi, Vietnam;

MNHN Muséum National d’Histoire Naturelle, Paris, France;

TBU PAR Faculty of Biology and Chemistry, Tay Bac University, Son La Province, Vietnam;

QSMI Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand;

CIB Museum of Herpetology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China.

Molecular data were generated for the specimen reported herein from Yen Bai Province, Vietnam. Homologous sequences were obtained from GenBank. DNA was extracted using the standard salt-extraction method (Aljanabi and Martinez 1997), combined with lysis by proteinase K. The cytochrome c oxidase subunit 1 (COI) gene fragment (660 bp) was amplified using the primer pair UTF 5'-TGT AAA ACG ACG GCC AGT TCT CAA CCA AYC AYA ARG AYA TYG G-3' and UTR 5'-CAG GAA ACA GCT ATG ACT ARA CTT CTG GRT GKC CRA ARA AYC A-3', following the protocol of Lissovsky et al. (2010). PCR products were cleaned by elution with a concentrated saline solution from 6% polyacrylamide gel. Sequencing was performed using an ABI 3500 automatic sequencer (Applied Biosystems) and BigDyeTerminator 3.1 kits 103 (Applied Biosystems). The nucleotide sequence was aligned with the BioEdit software (Hall 1999) and further edited manually. The final sequences were deposited in GenBank (MW542529).

We combined the sequence of the new specimen of Parafimbrios reported here with nine sequences downloaded from GenBank. We selected two outgroups, Xenopeltis unicolor Reinwardt, 1827 AB179620, Acrochordus granulatus Schneider, 1799 AB177879 (Dong and Kumazawa 2005), and five species for our ingroup, P. vietnamensis Ziegler, Ngo, Pham, Nguyen, Le & Nguyen, 2018 MH884515 (Ziegler et al. 2018), P. lao Teynié, David, Lottier, Le & Vidal, 2015 KT374005 (Nguyen et al. 2015), KP410746, Fimbrios klossi Smith, 1921 KP410744–45, Xenodermus javanicus Reinhardt, 1836 KP410747 (Teynié et al. 2015), and Achalinus spinalis Peters, 1869 MK064591 (Peng et al. 2017; Li et al. 2020) of the family Xenodermidae (Table 2). We used MEGA v. 7.0. (Kumar et al. 2016) for phylogenetic analyses using the Maximum Likelihood (ML) method. The HKY+G+I model was selected as the most appropriate DNA substitution model for the dataset using jModelTest 2.1.10 (Posada 2008). Minimum evolution (ME) and neighbor-joining (NJ) analyses were used as a complement to our maximum likelihood analyses. Node support was estimated using 1000 bootstrap replicates. Lastly, we calculated uncorrected pairwise divergences between all samples in MEGA v. 7.0.

Morphological characters of the second male specimen are concordant with those in the original description of Parafimbrios vietnamensis by Ziegler et al. (2018). The specimen has a cylindrical and slender body (Figs 2, 3). Head not distinct from neck, dorsally covered with large shields; eyes middle-sized, with a vertically sub-elliptic pupil.

Figure 2. 

Parafimbrios vietnamensis ZISP 31426 A dorsal view in life B ventrolateral view in life C dorsal view in life D ventral view of the preserved specimen.

Snout-vent length 298 mm; tail length 56 mm; total length 354 mm; ratio of tail length to total length 0.163. Dorsal scale rows 31-35-27; laterally rounded ventral scales 172+2; subcaudals 48; postoculars 2/2; preoculars 1/1; suboculars 1/1; supralabials 8/8; infralabials 7/7 (Fig. 3A, B). Rostral triangular, its upper edge separating it from the internasals; nasal in contact zone with rostral with curved raised edge; suture between the internasals much longer than that between the prefrontals; 8 supralabials, the first four bearing raised edges; temporals 2+3/2+3 (Fig. 3A, B); 48 unpaired subcaudals; total length 354 mm; tail length 56 mm; TaL/TL ratio 0.163.

Figure 3. 

Parafimbrios vietnamensis ZISP 31426 in life A dorsal view of head B ventral view of head C dorsolateral view of midbody D ventrolateral view of midbody.

Dorsal scales small, cycloid, keeled from region behind the neck onwards, every second scale of outermost row distinctly enlarged; 31 scales around the anterior part of the body; two dorsal scale rows corresponding to a ventral plate; distinct, laterally-rounded ventrals; single subcaudal scales; cloacal shield entire (Figs 2D, 3C, D). Left hemipenis basally everted.

Morphological data of this new specimen, the holotype of P. vietnamensis, and comparative data on seven specimens of P. lao are summarized in Table 1. The second male specimen from Vietnam differs from the male holotype of P. vietnamensis in having a larger size (SVL 298 mm vs. 222 mm), a higher number of ventrals (172+2 vs. 164), and fewer temporals (2+3/2+3 vs. 4+4/4+5). The pholidosis characteristics of the number of ventrals and temporals look more similar to those seen in P. lao.

Phylogenetic analysis. Molecular analyses corroborate the morphological data. The new specimen is significantly genetically divergent from P. lao by at least 10%. The new sample was strongly supported as the sister lineage to the holotype of Parafimbrios vietnamensis (bootstrap support = 100%) (Fig. 5). Genetic divergence between the newly-collected specimen and the holotype was low (1.7%), and thus supports that they are conspecific. The mean uncorrected pairwise genetic distance between the two species within the genus Parafimbrios was 7.7±0.8%.

Coloration. The color of the dorsum is gray-brown, with varying intensity of brown depending on the angle of the light, and with iridizing sequins. The head is separated from the neck by a wide, light-colored nuchal collar extending to the ventral surface and to the chin; the collar does not completely cover the ventral part of the chin but stops at the ventral scales. The dorsal surface of the head is reddish-brown from the lateral edge of the head to the parietal scale and frontal scales. The gular region is brown; the two huge mandibular plates are brown anteriorly, lightening posteriorly. The color of the nuchal collar is a light cream, slightly white-pinkish. The color of the belly is smoky gray with lightened lateral edges of the abdominal scales.

Distribution. Parafimbrios vietnamensis is only known from two provinces (Lai Chau Province, Yen Bai Province) in northwestern Vietnam (Fig. 1). The second species, P. lao, is only known from northern Vietnam in the Son La Province, but has a much wider distribution in Laos, Thailand, Vietnam and southern China. This area of Vietnam is the highland region associated with Hoang Lien Son ridge (Lai Chau, Lao Cai, and part of Yen Bai). Biogeographically, the species inhabits the south-eastern part of the Sikang-Yunnan floristic Province of the Holarctic floristic kingdom (Takhtajan 1978).

Ecology and habitat. The specimen was collected on November 30, 2019 after an overnight rain, at midnight. The specimen was found in the leaf litter in primary polydominant forest (Fig. 4) at an elevation of 1300 m a.s.l.

Figure 4. 

Habitat of P. vietnamensis in the primary polydominant forest in Yen Bai Province, Vietnam.

The second male specimen from Vietnam differs from the male holotype of P. vietnamensis in having a larger size (SVL 298 mm vs. 222 mm), a higher number of ventrals (172+2 vs. 164), and fewer temporals (2+3/2+3 vs. 4+4/4+5). The number of ventrals and temporals are similar to those reported from P. lao. Morphological data from our new finding, the holotype of P. vietnamensis, and comparative data for seven specimens of P. lao are summarized in Table 1.

Due to the only minor morphological differences between the holotype of Parafimbrios vietnamensis and the new specimen described in this paper, and to the very low genetic divergence, we consider the new specimen conspecific with the holotype of P. vietnamensis. The expanded diagnosis of the species is as follows:

A species of the genus Parafimbrios, characterized by the following combination of characters: 1) rostral laterally with two raised, curved edges; the upper one, together with a horizontal curved ridge of tissue, separates the rostral from the internasals; 2) nasal in contact zone with rostral with curved, raised edge; 3) nasal in contact zone with supralabials with two small oblique, curved raised edges located above first and second as well as above second and third supralabials; 4) suture between the internasals much longer than that between the prefrontals; 5) supralabials 8, the first four bearing raised edges; 6) infralabials 7; mental and anterior three to four infralabials with raised edges; 7) temporals 2+3.2+3–4+4–5; 8) (31–35)-(33–35)-(27–29) dorsal scale rows; 9) laterally rounded ventrals 164–172+2; 10) unpaired subcaudals 48–49; 11) total length at least 266–354 mm in males (with a tail length of 44–56 mm and a TaL/TL ratio of 0.16). The coloration corresponds to that given in the original description (Ziegler et al. 2018): brownish-black above, with a broad yellow neckband widening towards the venter and stretching to the chin region; dorsal head surface in part reddish-brown, in particular in the middle of the parietals and frontal; venter grayish-brown, lighter anteriorly, darker towards tail region; ventrals anteriorly and laterally darker. It should be noted that we are most likely dealing with subadult specimens, with a pronounced whitish-pinkish nuchal collar. For example, in the related species Parafimbrios lao, the nuchal collar dims and disappears with age, which can be seen in Teynié et al. (2015: fig. 4). More findings of this rare snake will add information to this species’ diagnosis.

Parafimbrios vietnamensis was previously known only from Hoang Ho Village, Phang So Lin Commune, Sin Ho District, Lai Chau Province in northern Vietnam (Ziegler et al. 2018). Although the new locality in Yen Bai Province is more than 200 km southeast of the type locality, the samples exhibit a relatively low amount of genetic divergence from each other.

Figure 5. 

ML phylogenetic tree (HKY+G+I model) based on the mitochondrial COI gene. Numbers near the branches denote percentage bootstrap resampling support from 1000 replications (ML/ME/NJ). Bootstrap support is only shown for values exceeding 80%. Asterisks represent 100% values. Scale bar shows substitutions/site.

The mountain systems in this area are composed of magmatic silicate rocks, particularly granite and quartzite, which were formed as extensive intrusions of late Paleozoic and Mesozoic age (Dovzikov et al. 1965). Tertiary tectonic movements uplifted these mountain terrains up to the present-day elevations, and subsequent erosion processes formed the present-day characteristic rocky landscape of this highland area, with very steep slopes, numerous rocky cliffs and deep and narrow river canyons (Fridland 1961; Averyanov et al. 2003). This area is extremely humid, with warm rainy summers and cold foggy and misty winters and without a distinct dry period. The peak of rainfall arrives in the summer months. Morning dew is very common throughout the mountain zone, as well as frequent heavy fog. Humid, cold, northeast monsoon winds that bring heavy fog, mist and drizzle are very common in the winter and early spring (Averyanov et al. 2003). Zonal types of vegetation in the studied area belong to a group of closed evergreen tropical monsoon (seasonal) submontane forests (Averyanov et al. 2003).

There are now three known specimens of genus Parafimbrios recorded in Vietnam: one of P. lao and two of P. vietnamensis. The discovery highlights the difficulty of finding specimens of Xenodermidae and the need for further surveys in these areas of Vietnam and Eastern Laos.

So far, the following snake species were reported from Che Tao Village, Che Tao Commune, Mu Cang Chai District, Yen Bai Province in Vietnam: Oreocryptophis porphyraceus (Cantor, 1839), Hebius bitaeniatus (Wall, 1925), H. boulengeri (Gressitt, 1937), Pararhabdophis chapaensis Bourret, 1934, Sinonatrix percarinata (Boulenger, 1899), Pareas hamptoni (Boulenger, 1905) (Le et al. 2018). This new record of the rare snake species Parafimbrios vietnamensis is an important supplement to the list of snakes recorded from Yen Bai Province, and highlights its conservation needs.