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A NEW SPECIES OF KRAIT (SQUAMATA: ELAPIDAE) FROM THE RED RIVER SYSTEM OF NORTHERN VIETNAM
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Ulrich Kuch, David Kizirian, Nguyen Quang Truong, Robin Lawson,Maureen A. Donnelly, And Dietrich Mebs |
We describe a new species of krait (Elapidae: Bungarus) from the Red River drainage in northern Vietnam. The new species differs from all congeners except its sister species Bungarus bungaroides by the combination of divided subcaudals, dorsal scales arranged in 15 rows, black and white rings on body and tail, and in color pattern of the head as well as hemipenis morphology. The new species differs from B. bungaroides , a distantly allopatric species ranging from eastern Nepal to northern Myanmar, in molecular characters and color pattern. We propose a vicariance hypothesis in which speciation coincided with the uplift of intervening mountain ranges in southwestern Yunnan (China) and/or Late Tertiary glaciations.
ELAPID snakes of the genus Bungarus Daudin,1803-commonly known as kraits-inhabit most of the Oriental faunal region with the exception of the Philippines. Throughout their range, these nocturnal and largely ophiophagous active predators are among the most dangerous and medically important venomous snakes (Warrell, 1999). Twelve species of kraits are currently recognized (Slowinski, 1994; David and Ineich, 1999), the majority of which are rare in collections and almost entirely unknown ecologically and toxinologically.
One of the rarer kraits is Bungarus bungaroides (Cantor, 1839), which was described from ‘‘Chirra Punji’’ in the Khasi Hills of northeastern India (currently Cherrapunji, Meghalaya State, India) and had long been known from the single type only (Gu¨ nther, 1864; Fayrer, 1872). Only 12 additional specimens of B. bungaroides have been reported in the literature: five specimens from Sikkim (India), Labdah near Kurseong and ‘‘Darjeeling’’ (currently West Bengal State, India) by Blanford (1879) and Sclater (1891; see also Wall, 1924); two from northern Cachar District (Assam, India) by Annandale (1904); two collected by Kaulback’s expedition to extreme northern Myanmar (Smith, 1940); one specimen each at two localities in Ilam District of eastern Nepal (Shah, 1999; Kabisch, 2002); one from the Brahmaputra River in the valley of Yarlung Zangbo Jiang (Medog County, southeastern Tibet; Rao, 2000); and one in Nagaland (northeastern India; Ao et al., 2004). Collectively, these records suggest an eastern Himalayan distribution for B. bungaroides (Fig. 1) with elevational records ranging from 250 m (Ao et al., 2004) to about 2100 m (Blanford, 1879; Boulenger, 1890).
Bungarus bungaroides is distinguished from all congeners by a combination of characters that includes divided subcaudals, dorsal scales arranged in 15 rows, a clear zone of demarcation between the calyculate and spinose zones of the hemipenis, an elongate choanal process of the palatine, the lack of expanded postzygapophysial processes, wide black and narrow white rings on body and tail, and a pattern of light lines and spots on the snout and sides of the head, rendering this species one of the morphologically most distinctive kraits (Slowinski, 1994).
During biotic inventories in northern Vietnam, specimens referable to B. bungaroides were collected in the “Song Hong”, or Red River, drainage in Yen Bai and Lao Cai Provinces, nearly 1000 km southeast of the closest known locality of this species. Despite their overall similarity to Indian B. bungaroides, the Vietnamese material exhibited notable differences in color pattern (e.g., Kizirian et al., 2002). Those phenotypic differences and the absence of B. bungaroides from vast intervening mountain regions such as Yunnan Province, China (Zhao and Adler, 1993) or Laos (Stuart, 1999) prompted us to further investigate the relationships and taxonomic status of the Vietnamese material.
Here we report evidence that reveals significant genetic differentiation between the distantly allopatric specimens from Vietnam and B. bungaroides (auct.) and supports the monophyly of mitochondrial lineages from four populations of B. bungaroides in India, Tibet, and Myanmar with respect to the Vietnamese lineage. In addition, we identify characters of the external phenotype that are constant in known individuals of B. bungaroides and serve to distinguish this species from the Red River kraits. Consequently, employing the criteria of apomorphy and diagnosability (sensu de Queiroz, 1998, 1999), we propose recognition of a new species of Bungarus from Vietnam.
MATERIALS AND METHODS
Fieldwork. Individuals of the new species were collected at night in (Kizirian et al., 2002) or near streams. Latitude, longitude, and elevation were determined using a GarminH GPS 48 personal navigator. After removal of tissue samples, specimens were fixed in formalin and then transferred to 70% ethanol for permanent storage.
Museum specimens. We attempted to locate specimens of B. bungaroides in collections worldwide and received information on 12 existing specimens. We examined six of these, including the single type specimen. Data on selected characters of the external phenotype of another specimen were provided by K. B. Shah (NHM). In addition, UK examined preserved museum specimens of all other recognized taxa of Bungarus, including larger series of wide-ranging species. Museum acronyms include those in Leviton et al. (1985) and IEBR (Institute of Ecology and Biological Resources, National Center for Natural Science and Technology of Vietnam, Hanoi, Vietnam), KIZ (Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China), and NHM (Natural History Museum, Tribhuvan University, Kathmandu, Nepal).
Morphology. Specimens of the new species were compared to specimens of all recognized taxa of Bungarus. Figures were drawn using a camera lucida. We used the scalation terminology of Campbell and Lamar (2004) and the ventral scale count method of Dowling (1951). Dorsal scale rows, where given as three values, were counted one head length behind the angle of the jaw, at midbody, and one head length before the cloaca. Dorsal scale row reductions on the neck
were recorded by counting scale rows from the first row diagonally toward the head up to the vertebral scale, and then again down to the first row on the other side of the body. Dorsal scaleb row reductions on the tail were recorded in an analogous manner, but oriented toward the tail tip. The following straight-line measurements were made using dial vernier calipers to the nearest 0.1 mm: head length (distance between angle of jaw and snout-tip), maximal head width,
head width at the level of the eyes (distance between anterior external margins of supraoculars), internarial distance, and maximal eye diameter. Head scale measurements were made in an analogous manner; comparisons of scale proportions are based on maximum straight-line distances unless indicated differently. A string was used to determine the snout-vent length (from tip of snout to posterior margin of anal plate) and the tail length (from posterior edge of
anal plate to tip of tail). Bilateral variation is reported as left/right. In color descriptions, the capitalized colors and color codes (the latter in parentheses) are those of Smithe (1975).
DNA extraction and sequencing.-Samples of liver and muscle tissue were taken from the holotype of the new species (IEBR 1172 [LACM FS 843]) using sterilized dissecting tools immediately after the administration of a lethal dose of sodium pentobarbital on 2 October 2001. Tissues were stored in DMSO buffer (20%), maintained at ambient temperature while in the field, and later stored at 270 C (LACM) and 4 C (IEBR). Tissue samples were also obtained from Rao’s (2000) ethanol-preserved specimen of B. bungaroides (KIZ 98R0186; Table 1) and were stored in 95% ethanol. Small amounts of subcutaneous muscle tissue were removed from areas adjacent to existing incisions in four historical museum
specimens of B. bungaroides with an unknown history of preservation, originating from ‘‘Darjeeling,’’ India (BMNH 80.11.10.140, BMNH 1940.3.9.11) and northern Myanmar (BMNH 1940.6.5.61, BMNH 1940.6.5.62). Tissue samples from these specimens were transferred to individual tubes and maintained at ambient temperature in aliquots of the preservative fluid of the corresponding museum specimen until use for DNA isolation.
Tissue samples from the holotype of the new species (IEBR 1172) and from the B. bungaroides of Rao (2000) were subjected to standard proteinase K digestion (Sambrook and Russell, 2001). For the isolation of DNA from tissue samples of the historical specimens, we (UK) used a modification of the protocol by Chatigny
(2000), which combines a prolonged proteinase K digestion with chemical treatment to break disulfide bonds in proteins. These tissue samples were washed twice with 1 ml 70% ethanol, each time followed by brief shaking and 10 min centrifugation at 13,000 rpm. Aliquots of tissue were then cut into small pieces on blotting paper, transferred to a 1.5-ml cryovial, which was left open to dry for 5min at room temperature. After adding 1 ml of sterile water, the samples were shaken and left to re-hydrate for 20 min at room temperature, followed by 15 min centrifugation at 13,000 rpm. Then, water was removed and 1 ml of buffer (0.1 M NaCl; 0.05 M Tris-HCl, pH 7.5; 0.001 M EDTA) was added along with 25 ml of proteinase K (20 mg/ml), 50 ml of 20% sodium dodecyl sulfate (SDS), and 2.5 ml of 1 M dithiothreitol
TABLE 1. DDBJ/EMBL/GENBANK ACCESSION NUMBERS OF NUCLEOTIDE SEQUENCES OBTAINED IN THIS STUDY.
Taxon
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Locality |
Catalog number |
Tissue number |
cytb |
ND4 |
B. bungaroides |
‘‘Ahke, Triangle Upper Burma’’[Ahke, Kachin, Myanmar] |
BMNH 1940.6.5.61
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- |
AJ748688
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AJ830217
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| B. bungaroides |
‘‘Matsatap,Triangle Upper Burma’’[Matsatap, Kachin,
Myanmar] |
BMNH 1940.6.5.62
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- |
AJ748687 |
AJ830216 |
| B. bungaroides\ |
Medog, Medog County, Xizang, China (Brahmaputra, Tibet) |
KIZ 98R0186 |
- |
AY973270
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- |
| B. bungaroides |
‘‘Darjeeling’’[Darjiling,
West Bengal, India] |
BMNH
1940.3.9.11
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- |
AJ748689
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AJ830218
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| B. n. sp. |
Na Hau Commune,
Van Yen District, Yen Bai,
Vietnam (Red River,
Vietnam) |
IEBR 1172
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LACM FS 843
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AJ749306 AJ830250
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AJ830716
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| B. candidus |
Purwokerto, Central Java,
Indonesia |
- |
UK-BP4
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- |
AJ830231
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| B. multicinctus |
Taiwan |
- |
UK-Bm9204
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- |
AJ830249
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(DTT). The vials were inverted twice and incubated in a gently shaking water bath for 24 h at 55 C to complete macroscopic lysis of the tissue. An additional 25 ml of proteinase K, 25 ml SDS, and 2.5 ml DTT were added, followed by 20 h incubation at 55 C. Lysates of all tissue samples were subjected to two phenol-chloroform-isoamyl alcohol and two chloroform-isoamyl alcohol extractions. DNA was precipitated with 1 ml icecold 98% ethanol and 50 ml 3 M sodium acetate (pH 4.5) at 220 C overnight. Samples were then centrifuged for 30 min (DNA from IEBR 1172) or 1.5 h (BMNH samples) at 13,000 rpm and 4 C and the supernatant removed using a micropipette. Samples were washed twice with 500 ml ethanol (70%), followed each time by 10 min centrifugation at 13,000 rpm and 4 C and removal of the supernatant, except for approximately 20 ml at the bottom of the tube which were removed by evaporation in a heat block (50 C). The precipitated DNA was dissolved in sterile water at 4 C overnight. This stock solution, as well as 1:10, 1:100, and 1:1000 dilutions, were used for PCR.
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