Larval morphology in four species of Hylarana from Vietnam and Thailand with comments on the taxonomy of H. nigrovittata sensu latu (Anura: Ranidae) Author Gawor, Anna Author Hendrix, Ralf Author Vences, Miguel Author Böhme, Wolfgang Author Ziegler, Thomas text Zootaxa 2009 2051 1 25 journal article 10.5281/zenodo.186587 f979f9ce-284c-46d4-a6b0-25dfc8f77007 1175-5326 186587 Hylarana nigrovittata ( Blyth, 1856 ) from central Vietnam Series examined. Tadpoles of Hylarana nigrovittata (ZFMK 87279: n = 21) were collected in PNKB, Khe Van region, Vietnam by R. Hendrix in July 2006 . The larvae were collected throughout day and night time in a primary forest stream at an altitude of about 130 m above sea level. TABLE 7. Measurements (min.-max., mean and standard deviation, in mm) of the adult Hylarana nigrovittata series from Phong Nha – Ke Bang National Park, Quang Binh Province, Vietnam (ZFMK 71298-71300); for abbreviations see material and methods. Measurements Males (n = 3) SVL 37.5 – 43.0 (39.50 ± 3.04) SL 7.0 – 7.5 (7.17 ± 0.29) NL 3.0 – 3.5 (3.33 ± 0.29) EN 4.0 – 4.5 (4.17 ± 0.29) HW 14.5 – 17.0 (15.50 ± 1.32) HL 14.0 – 16.0 (15.00 ± 1.00) EE 8.5 – 9.0 (8.83 ± 0.29) ED 5.5 TD 4.0 – 4.5 (4.30 ± 0.26) HaL 12.0 – 12.5 (12.33 ± 0.29) LL 68.0 – 73.0 (71.00 ± 2.65) FL 22.0 – 23.5 (22.50 ± 0.87) IMT 2.0 – 2.5 (2.23 ± 0.25) Identification. Adult H. nigrovittata specimen (ZFMK 71298-71300) from Phong Nha - Ke Bang, Quang Binh Province, Vietnam ( Fig. 1d ; for measurements see Table 7 ) largely agreed with the description provided by Inger et al. (1999) and Ziegler (2002) . Sequence congruence between syntopically sampled adult specimens and tadpoles (ZFMK 87279a–d) was 99.8 %. The following larval description is based on a single tadpole in Gosner stages 35/36 ( Fig. 6 ; for measurements see Table 8 ). The tadpole specimen used for description and drawing originates from the same water body as the DNA voucher specimen (characters of the DNA voucher specimen of series ZFMK 87279: stage 29, BL 8.5 mm , NP 52, NK 35 per 0.5 mm ). In this water described specimen is therefore body, also H. guentheri (adults only) was found. The DNA voucher specimens fully agree morphologically with the described and drawn specimen, including the characters that diagnose H. nigrovittata from H. guentheri (narrow upper jaw sheath, finger-shaped marginal papillae, one submarginal row of papillae, papillae pigmented at the basis, reddish to olive brown marbled colour with light blotches on tail). The identification ofwell supported. Colour pattern. Colouration of tadpole in preservative: body marbled, dorsally and laterally covered with brownish to olive-coloured blotches. Pigmentation is strongest around the nares, in the anterior and posterior corners of the eyes, and in the region of the intestinal coils. Single green pigments can be found around the mouth and on the throat region. Chest and belly white to transparent, without pigmentation, intestinal spiral well visible in ventral view. Tail marbled with brown to green pigments. Musculature of tail is white to yellow, upper and lower fin transparent. Hind limbs yellow to white, dorsolaterally with pigments. Colouration in life: body marbled, dorsally and laterally with drab, olive-coloured to brown blotches. Ventral side of body olive-coloured, slightly transparent. Throat and belly silvery. Musculature and lower fin of tail grey to olive with light-coloured blotches. Upper fin of tail is transparent. Description in dorsal view: Body shape oval, elongated (maximum body width 0.60 of body length), with a rounded snout. Eyes (maximum eye diameter 0.11 of body length) directed and positioned dorsolaterally, at the end of first body third, not visible in ventral view (interpupilar distance 0.57 of maximum body width). Nares small and rounded, anterodorsally positioned (rostro-narial distance 0.56 of naro-pupilar distance). Internarial distance 0.63 of interpupilar distance. Tail musculature well developed (width of tail musculature at base 0.49 of maximum body width). FIGURE 6. Drawings of the preserved tadpole of Hylarana nigrovittata (stages 35/36) from Phong Nha – Ke Bang National Park, Quang Binh Province, Vietnam; (A) dorsal view, (B) lateral view, (C) oral disc. Drawings by Anna Gawor Description in lateral view: Body depressed (maximum body height 0.69 of maximum body width). Snout rounded. Spiracle sinister, laterally positioned (distance from snout tip to opening of spiracle 0.67 of body length), conical-shaped, oriented in posterodorsal direction and slightly visible in dorsal view. Tail length 1.78 of body length. Musculature of tail well developed (height of tail musculature at base 0.47 of maximum body height, and 0.45 of maximum tail height). Tail musculature runs parallel, tapering from the base to the middle of tail fin, thereafter continuously tapering towards the tip of the tail. Upper fin situated at base of tail, slightly convex (maximum height of upper fin 0.34 of maximum tail height). Maximum height of lower fin 0.60 of maximum height of upper tail fin. Vent tube medially located at body end, ventrally positioned, cloacal aperture dextral in dorsal view and not adhered to lower tail fin. Oral disc. Oral disc anteroventrally positioned, laterally emarginated (oral disc width 0.38 of maximum body width). Oral disc framed by finger-shaped, pigmented papillae except for a large medial gap on the upper labium. All papillae show a pointed end and are transparent. Marginal papillae of the upper labium elongated. Few submarginal papillae situated on each side between upper and lower labium in the corner of mouth. In addition there is a row of submarginal papillae at the lower labium under the last keratodont row. In total 54 papillae. Keratodont row formula 2(2)/3(1), with 28 keratodonts per 0.5 mm . Jaw sheath black, and lightly serrated. Upper jaw sheath curved with large appendices, lower jaw sheath V-shaped. TABLE 8. Measurements (min.-max., mean and standard deviation, in mm) of the Hylarana nigrovittata larvae series from Phong Nha – Ke Bang National Park, Quang Binh Province, Vietnam (ZFMK 87279 n=21); for abbreviations see material and methods.

Stage BH	BL BW	ED	IND	IP LF	MTH	NK	NP
1.3–1.4	3.4–3.7 2.3–2.6	-	1.1–1.4 0.3–0.4	1.2–1.6	-	19–25
24 (1. 3 7 ±0.06)	(3. 5 2 (2. 4 2 ±0.18) ±0.13)	0.4	-	(1. 2 2 (0. 3 0 ±0.13) ±0.05)	(1. 3 3 ±0.19)	-	(22 ±3)
(n=3)	(n=3) (n=3)	(n=3)	-	(n=3) (n=3)	(n=3)	-	(n=3)
1.5–2.0	4.0–4.5 2.8–3.1	-	1.5–1.7 0.2–0.4	1.3–1.4	-	36–39
25 (1. 7 5 ±0.35)	(4. 2 5 (2. 9 5 ±0.35) ±0.21)	0.5	-	(1. 6 0 (0. 3 0 ±0.14) ±0.14)	(1. 3 5 ±0.07)	-	(38 ±2)
(n=2)	(n=2) (n=2)	(n=2)	-	(n=2) (n=2)	(n=2)	-	(n=2)
2.1–2.3	6.2–7.2 3.9–4.7	0.7–0.9	1.5–1.9	2.3–2.7 0.6–0.9	2.6–3.3	31–40	42–45
27 (2. 1 8 ±0.11)	(6. 7 0 (4. 2 8 ±0.71) ±0.60)	(0. 8 0 ±0.14)	(1. 7 2 ±0.26)	(2. 4 9 (0. 7 3 ±0.30) ±0.25)	(2. 9 3 ±0.53)	(36 ±6)	(44 ±2)
(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2)
29 2.7	8.5 4.6	1.1	2.0	2.8 -	-	35	52
2.2–2.8	6.0–7.1 3.4–4.7	0.7–0.8	0.9–1.7	1.8–2.5 0.6–0.8	2.5–3.2	33–36	40–55
30/31 (2. 4 8 ±0.39)	(6. 5 5 (4. 0 3 ±0.78) ±0.95)	(0. 7 5 ±0.07)	(1. 3 3 ±0.53)	(2. 1 3 (0. 7 0 ±0.53) ±0.49)	(2. 8 5 ±0.49)	(35 ±2)	(48 ±11)
(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2)
4.2–4.3	10.2–10.6 6.0–6.1	1.1–1.2	3.3–3.5 0.9–1.3	4.3–4.4	28–29	48–54
35/36 (4. 2 5 ±0.07)	(1 0. 40 (6. 0 5 ±0.28) ±0.07)	(1. 1 5 ±0.07)	2.2	(3. 4 0 (1. 1 0 ±0.14) ±0.28)	(4. 3 7 ±0.10)	(29 ±1)	(51 ±4)
(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2)
4.0–4.2	10.2–10.7 6.2–6.5	1.3–1.4	2.0–2.3	3.2–3.7 1.1–1.2	4.0–5.1	29–31	64–65
38/39 (4. 1 0 ±0.14)	(1 0. 45 (6. 3 5 ±0.35) ±0.21)	(1. 3 5 ±0.07)	(2. 1 5 ±0.21)	(3. 4 5 (1. 1 5 ±0.35) ±0.07)	(4. 5 5 ±0.78)	(30 ±1)	(65 ±1)
(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2)
4.0–4.1	10.5–11.1 6.1–6.2	1.3–1.4	2.2–2.3	3.7–3.9 1.0–1.2	4.4–4.7	30–34	59–63
40 (4. 0 5 ±0.07)	(1 0. 80 (6. 1 5 ±0.42) ±0.07)	(1. 3 5 ±0.07)	(2. 2 5 ±0.07)	(3. 8 0 (1. 1 0 ±0.14) ±0.14)	(4. 5 5 ±0.21)	(32 ±3)	(61 ±3)
(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2) (n=2)	(n=2)	(n=2)	(n=2)
4.0–4.4	10.8–11.2 6.4–6.8	1.3–1.7	2.0–2.2	3.7–3.90 0.8–1.3	3.4–5.1	29–35	57–69
41 (4. 2 0 ±0.20)	(11. 0 0 (6. 5 8 ±0.16) ±0.15)	(1. 4 8 ±0.15)	(2. 1 2 ±0.08)	(3. 7 8 (1. 0 4 ±0.08) ±0.18)	(4. 4 4 ±0.67)	(32 ±2)	(62 ±5)
(n=5)	(n=5) (n=5)	(n=5)	(n=5)	(n=5) (n=5)	(n=5)	(n=5)	(n=5)

continued.

Stage NPD	ODW	RND	SS	TAL	TL	TMH	TMW	UF
-	0.9–1.0	-	2.7–2.8	4.6–5.3	7.9–8.9	0.6–0.8	0.6–0.7	0.4–0.6
24 -	(0.9 2 ±0.08)	-	(2. 72 ±0.08)	(5. 0 3 ±0.38)	(8. 5 5 ±0.52)	(0. 7 2 ±0.08)	(0. 65 ±0.05)	(0. 5 0 ±0.05)

(n=3) - (n=3) (n=3) (n=3) (n=3) (n=3) (n=3)

-	1.2–1.3	- 3.0–3.2	0.8–0.9	0.7–0.9
25 -	(1.2 3 ±0.04)	- (3.08 ±0.11) 6.0	10.5 (0. 8 5 ±0.07)	(0.78 ±0.11) 0.5
-	(n=2)	- (n=2) (n=1)	(n=1) (n=2)	(n=2) (n=2)
1.2–1.3	1.6–2.1	0.7–0.8 4.6–5.5 9.9–10.7	16.9–17.1 1.4–1.5	1.3–1.9 0.9–1.0
27 (1. 2 5 ±0.07)	(1.8 5 ±0.35)	(0.73 ±0.11) (5. 0 5 (1 0. 3 0 ±0.64) ±0.57)	(1 7. 0 0 (1. 4 5 ±0.14) ±0.07)	(1. 60 (0. 9 8 ±0.42) ±0.04)
(n=2)	(n=2)	(n=2) (n=2) (n=2)	(n=2) (n=2)	(n=2) (n=2)
29 1.6	1.8	0.8 5.8 -	- 1.8	1.9 -
0.9–1.3	0.9–1.2	0.5–0.7 3.4–5.1 6.2–10.3	12.2–17.4 1.3–1.6	1.2–1.8 1.0–1.3
30/31 (1. 1 0 ±0.28)	(1.0 5 ±0.21)	(0. 6 0 (4. 25 (8. 2 5 ±0.14) ±1.20) ±2.90)	(1 4. 8 0 (1. 4 3 ±3.68) ±0.18)	(1. 48 (1. 1 3 ±0.46) ±0.18)
(n=2)	(n=2)	(n=2) (n=2) (n=2)	(n=2) (n=2)	(n=2) (n=2)

1.8–1.9 2.3–2.5	1.0–1.1	6.8–7.7	18.2–21.8 28.4–32.4
35/36 (1. 8 5 (2.4 0 ±0.07) ±0.14)	(1. 0 5 ±0.07)	(7. 25 ±0.64)	(2 0. 0 0 (3 0. 4 0 2.0 ±2.55) ±2.83)	3.0	1.5
(n=2) (n=2)	(n=2)	(n=2)	(n=2) (n=2) (n=2)	(n=2)	(n=2)
1.8–2.0 2.6–2.9	0.9–1.1	6.7–7.5	17.9–22.5 28.1–33.2 2.2–2.7	2.6–2.7	1.5–1.9
38/39 (1. 9 0 (2.7 5 ±0.14) ±0.21)	(1. 0 0 ±0.14)	(7. 10 ±0.57)	(2 0. 2 0 (3 0. 6 5 (2. 4 5 ±3.25) ±3.61) ±0.35)	(2. 65 ±0.07)	(1. 7 0 ±0.28)
(n=2) (n=2)	(n=2)	(n=2)	(n=2) (n=2) (n=2)	(n=2)	(n=2)
1.9–2.0 2.5–2.6	7.2–7.8	20.6–22.3 31.1–33.4 2.0–2.5	3.0–3.2	1.3–1.7
40 (1. 9 5 (2.5 5 ±0.07) ±0.07)	0.9	(7. 50 ±0.42)	(2 1. 4 5 (3 2. 2 5 (2. 2 5 ±1.20) ±1.63) ±0.35)	(3. 10 ±0.14)	(1. 5 0 ±0.28)
(n=2) (n=2)	(n=2)	(n=2)	(n=2) (n=2) (n=2)	(n=2)	(n=2)
2.0–2.3 2.4–3.0	0.8–1.2	7.7–8.2	20.5–22.5 31.3–33.7 2.0–2.8	2.7–3.5	1.0–2.0
41 (2.16 ±0.11) (2.6 2 ±0.24)	(0. 9 6 ±0.15)	(7. 94 ±0.23)	(2 1. 6 6 (3 2. 6 6 (2. 3 0 ±0.79) ±0.90) ±0.35)	(3. 14 ±0.29)	(1. 5 4 ±0.42)
(n=5) (n=5)	(n=5)	(n=5)	(n=5) (n=5) (n=5)	(n=5)	(n=5)

Glandular Zones. Secretory glands are discernible on each side of the body, situated dorsolaterally at midbody, laterally and posteroventrally at the end of body and ventrally under the mouth in the throat region. Measurements (in mm). BH 4.2; BL 10.2; BW 6.1; ED 1.1; IND 2.2; IP 3.5; LF 0.9; MTH 4.4; NK 28; NP 54; NPD 1.8; ODW 2.3; RND 1.0; TAL 18.2; TMH 2.0; TMW 3.0; UF 1.5. Variation within the series. Series with 21 tadpoles in stages 24–41. Proportions vary as follows: BW 0.54–0.70 of BL, ED 0.11–0.15 of BL, IP 0.43–0.63 of BW, RND 0.36–0.62 of NPD, IND 0.53–0.71 of IP, TMW 0.25–0.52 of BW, BH 0.45–0.72 of BW, TAL 1.03–2.10 of BL, TMH 0.45–0.71 of BH, TMH 0.45–0.67 of MTH, UF 0.24–0.42 of MTH, LF 0.40–1.10 times UF, ODW 0.19–0.47 times BW. In early developmental stages, the body skin is more transparent to white with only slight pigmentation, and the keratodont row formula is not definitive (only from stage 25 onwards). The intestinal spiral is always well visible in lateral and ventral view. Previous descriptions. In comparison to the data provided by Smith (1916) , Bourret (1942) and Manthey & Grossmann (1997) for larvae from Thailand , and compared with the data provided by Grosjean (2005) for Ben En National Park, southern Vietnam , we found the following differences: According to Smith (1916) the tail fin is about twice as large as the body. This corresponds with larvae in more advanced stages (34–41) from Phong Nha – Ke Bang, whereas the tail of younger larvae (stages 25–28) is only slightly longer than the body. The length of tail fins measured by Grosjean (2005) for H. nigrovittata is less than one and a half of body length across all stages of development. In addition, Grosjean (2005) mentions only four pairs of glandular zones which are located dorsally between the eyes, dorsolaterally at midbody, dorsolaterally at the body end in front of the tail insertion, and posteroventrally at the body end. In the larvae from Phong Nha – Ke Bang, we could observe an additional pair of glands that is located posteriorly to the mouth in the throat region. Bourret (1942) mentions three uninterrupted keratodont rows [2(2)/3] on the lower labium of H. nigrovitatta larvae, whereas the tadpoles from Phong Nha – Ke Bang have an inner interrupted row and two outer continuous keratodont rows [2(2)/3(1)] on the lower labium which agrees with descriptions by Smith (1916) and Grosjean (2005) . Manthey & Grossmann (1997) give keratodont row formulae of 2(2)/3(1) or 3(2–3)/3(1). However, the latter formula and an additional row of interrupted keratodonts on the upper labium respectively was neither observed in our specimens nor by Grosjean (2005) . Natural history. Larvae of H. nigrovittata were collected during day time in a primary forest stream pool in the Khe Van area of Phong Nha – Ke Bang National Park, Vietnam , at 19 July 2006 . The stream pools were surrounded by larger rocks and measured up to 80 x 60 cm in diameter with water levels up to 10 cm . The larvae were found near the sandy ground which was completely covered by a thick layer of algae. During the night time (10:00 p.m.) of 19 July 2006 , air temperature was 27.2°C, relative humidity was 75%, water temperature was 27°C, and pH-value was 6.6. Larval densities were relatively high, with about 40 individuals in Gosner stages 25–41 in a pond of 60 x 40 cm diameter. Throughout day time, the larvae were hidden among the algae. The larvae were found motionless on the layer of algae and escaped into this layer when disturbed. Adult H. nigrovittata were regularly found in and near slowly running forest streams within the vegetation, under rocks and logs and in rock crevices up to 300 m a.s.l. Mating calls of males were regularly heard between 06:00 and 12:00 p.m. except for heavy rain falls. We once counted at least 30 calling males along 100 to 150 m forest stream length. Layers of eggs were seen several times on the water surface in former crab burrows at river banks of slowly running streams between 20 June 2006 and 0 6 August 2006 . The whitishblack yolk of eggs measured 1.5 mm in diameter and they always were found on the water surface.